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Proteomics/Protein Separations- Electrophoresis/Two Dimensional Polyacrylamide gel Electrophoresis(2D-PAGE)

2D-PAGE is a form of gel electrophoresis in which separation and identification of proteins in a sample are done by displacement in 2 dimensions oriented at right angles to one another(orthogonal). This technique is also used to compare two or more samples to find differences in their protein expressions.

Basis for separation
In this technique proteins are separated by two different physicochemical properties. In the first dimension proteins or polypeptides are separated on the basis of their net charges by isoelectric focusing and in the second dimension they are separated on the basis of their molecular masses by electrophoresis. Because it is unlikely that two molecules will be similar in both properties, molecules are more effectively separated in 2-D electrophoresis than in 1-D electrophoresis.

Sample preparation
The goal of sample preparation is to solubilize maximum number of proteins and maintain their solubility throughout the process. The materials for sample should be carefully collected, snap frozen and ground under liquid nitrogen in the presence of protease inhibitors. After extracting proteins from source material they are then solubilized and denatured by means of chaotropes, detergents, and reducing agents. Hydrogen-bonds in the sample proteins are disrupted by chaotropes urea and thiourea. Uncharged detergents are used to disrupt hydrophobic interactions. Detergents such as CHAPS, Triton X-100, sulfobetaine SB3-10, and amidosulfobetaine are IEF-compatible additives. Disulfide bonds are reduced to sulfhydryls by reducing agents dithiothrietol (DTT), dithioerythritol, and tributyl phosphine (TBP).

Sequential extraction is done to categorize proteins based on their solubility. This is an example of pre-fractionation to enrich low abundance proteins. Proteins are sequentially extracted into chaotrope/detergent solutions of increasing solubilization power. First, proteins are treated with an aqueous buffer, the insoluble proteins remaining from this extraction are treated with urea/CHAPS/TBP, and the insoluble proteins remaining from this step are then treated with urea/thiourea/CHAPS/SB 3-10/TBP. 2-D PAGE is then done to separate the proteins in each of the supernatants. The remaining insoluble material from the final extraction can be taken up in SDS-PAGE sample solution and run in a one-dimensional gel.

Isoelectric focusing (IEF)
In IEF, proteins are separated by electrophoresis in a pH gradient based on their isoelectric point(pl). A pH gradient is generated in the gel and an electric potential is applied across the gel. At all pHs other than their isoelectric point, proteins will be charged. If they are positively charged, they will move towards the more negative end of the gel and if they are negatively charged they will move towards the more positive end of the gel. At its isoelectric point, since the protein molecule carry no net charge it accumulates or focuses into a sharp band.

Immobilized pH Gradient (IPG) and IEF run
Immobilized pH gradients are used for IEF because the fixed pH gradients remain stable over extended run times at very high voltages. The pH gradients of IPGs are generated by means of buffering compounds that are covalently bound into polyacrylamide gels. IPGs are cast strips with plastic backing sheets and are commercially available in different pH ranges and lengths. They offer high resolution, great reproducibility, and allow high protein loads. Isoelectric focusing is run in the same solutions that are used to extract or solubalize the proteins. The IPG strips with the protein sample must be rehydrated in the rehydration/sample buffer during with protein samples are loaded into the strips. Rehydration can be active or passive. To load larger proteins active rehydration in small voltage is applied. After the run in IEF cell, the proteins focus as bands on the strip according to their isoelectric points. The focused strips can be frozen for storage

IPG gel strips equilibration
The proteins in the focussed IPG strips are uncharged because they are at their pI and so they will not move into the SDS- PAGE gel. So the strips are treated with SDS (sodium dodecyl sulfate), an anionic detergent which denatures the protein by breaking the disulfide bonds and gives negative charge to each protein in proportion to its mass. Without SDS, different proteins with similar molecular weights would migrate differently due to differences in folding, as differences in folding patterns would cause some proteins to better fit through the gel matrix than others. SDS linearizes the proteins so that they may be separated strictly by molecular weight. The SDS binds to the protein in a ratio of approximately 1.4 g SDS per 1.0 g protein (although binding ratios can vary from 1.1-2.2 g SDS/g protein), giving an approximately uniform mass:charge ratio for most proteins, so that the distance of migration through the gel can be assumed to be directly related to only the size of the protein. Proteins may be further treated with reducing agent, such as dithiothreitol (DTT) or TRP(Tributyl phosphine to break any reformed disulfide bonds and then alkalated with iodoacetamide to prevent reformation of disulfide bonds. A tracking dye like bromophenol blue may be added to the protein solution to track the progress of the protein solution through the gel during the electrophoretic run.

SDS-PAGE run
The equlibrated IPG strip is placed on the top of the SDS-PAGE gel submerged in a suitable buffer and sealed in place with agarose gel. An electric current is applied across the gel, causing the negatively-charged proteins move out of the gel and migrate across the gel. Depending on their size, each protein move differently through the gel matrix. Smaller proteins travel farther down the gel, while larger ones remain closer to the point of origin. The proteins separate roughly according to size and therefore by molecular weight. It is common to run "marker proteins" of known molecular weight in a separate lane in the gel, in order to calibrate the gel and determine the weight of unknown proteins by comparing the distance traveled relative to the marker.

Visualization
After electrophoresis the gel is stained to visualize the separated proteins. Commonly used stains are Coomassie Brilliant Blue or SYPRO Ruby or silver stain. different proteins will appear as distinct spot within the gel. Coomassie Brilliant Blue or SYPRO Ruby are compatible with Mass Spectrometry. Coomassie Brilliant Blue has detection limit about 10ng of proteins per spot and the gel images of spots can be captured by scanning densitometer which operate in visible light[]. SYPRO Ruby can detect 1ng of proteins per spot and since it is fluorescent, the sopts are visualized by a fluorescent imager. Silver stain can detect spots containing proteins less than 1 ng and is the most sensitive non – radioactive protein visualization method.Laser devices for image capturing are useful for fluorescently stained gels.

Analysis
The images can be further analyzed using image –analysis softwares. These softwares quantify proteins spots, match images and compare corresponding spots intensites of related gels, prepare gel data reports, remove background patterns, and integrate image information to databases.Alternately the proteins separated can be obtained from the gel can be analyzed by MS for protein identification. 2D- PAGE can be used to study differential protein expression by comparing images from 2D –PAGE gels samples labeled with stable isotopes or fluorescent dyes.

Advantages and Disadvantages
Using 2D-PAGE,100 –1000’s of polypeptides can be analyzed in a single run. The proteins can be separated in pure form from the spots. The spots can be quantified and also analyzed by MS. In this method polypeptides can be probed with antibodies and also they can be tested for post- translational modifications. The disadvantages may include large amount of sample handling, less reproducibility, difficulty to separate low abundance proteins, acidic and basic proteins, very large and very small proteins and hydrophobic proteins.Also not automated for high throughput analysis.2D-PAGE has limited dynamic range.


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Proteomics/Protein Separations- Electrophoresis/Differential in Gel Electrophoresis(DIGE)

Theory
Differential in Gel Electrophoresis (DIGE) is a technique to monitor the differences in proteomic profile between cells in different functional states. This is done in three steps. First, samples are tagged with unique fluorescent dyes. Second, they are run together on the same 2D-PAGE gel. Finally, after the run completes, the different fluorescent images of the same gel are superimposed over each other. DIGE allows the study of proteins that are expressed differentially, as well as those that are common between samples. This technology allows for simultaneous separation and comparison of up to three samples on one gel.

DIGE vs. 2D-PAGE
In traditional 2D-PAGE, different samples are often separated in multiple gels. Those gels are then overlapped to compare one gel to another. Due to differences between gels in spatial resolution and spot intensities, the overlaying of images and correct matching of proteins is difficult. There can also be variations in protein uptake by the isoelectric focusing strips, incomplete protein transfer from the first to the second dimension gel, and local inconsistencies in gel composition, field strength or pH gradients. These gel-to-gel variations can mask the biological variation between the samples. Unfortunately, not all of these variations are avoidable; they can occur for a number of reasons. Thus, quantitative comparisons of protein expression levels are difficult using 2D-PAGE.

In DIGE, protein mixtures are pre-labeled, prior to electrophoresis, with cyanine dyes that guarantee co-migration of proteins. This co-migration on the same gel eliminates running differences between samples. In addition, the samples are subjected to the same environment and the same procedures throughout the experiment. Experimental variation is minimized in this way. In DIGE, there is no need to process a gel for visualization after electrophoresis.

Dyes
The cyanine dyes (Cy2, Cy3 and Cy5) used for DIGE are N-hydroxy succinimidyl ester derivatives, are covalently tagged to the ε-amino group of lysine residue of proteins, and replace the ε-amino group positive charge with the positive charge of the dye. The binding of the dyes introduce small but matched increases in molecular weight to the protein. Since these dyes are hydrophobic, to prevent precipitation of proteins, only one lysine residue per protein is labeled (minimal labeling). Minimal labeling limits the fluorescence intensity and thus the sensitivity of the stain. The dyes are all charge-matched and molecular mass-matched to prevent alterations of the isoelectric point, and to minimize dye-induced shifting of labeled proteins during electrophoresis.

There is also another class of cyanine dyes, called saturation dyes, which saturate cysteine residues instead of minimally labeling lysine residues. Again, these dyes are mass and charge-matched; but there is a smaller chance of protein modification and isoelectric point shift. Saturation cysteine dyes have superior sensitivity to minimal lysine labeling. As with the minimal lysine labelling cyanine dyes, if a cysteine amino acid is not present in a protein, then the saturation dyes will not be able to label it, leaving the protein undetectable.

Two dyes
Two protein samples are first labeled with propyl-Cy3 and methyl-Cy5. Once the samples are labeled, they are applied onto a single 2D-GE gel. After the run, the corresponding protein spot are visualized by successively illuminating the gel with the excitation wavelengths of each of the dyes. The resulting protein spots are analyzed using imaging software. Differentially expressed protein spots are then excised from the gel and identified by mass spectrometry.

Three dyes
To study multiple samples, a large number of pairwise comparisons or analysis of samples run on different gels is needed. An important alternative is to use a third dye (Cy2) with similar characteristics as Cy3 and Cy5. Protein samples are labeled with either Cy3 or Cy5, and a pooled sample containing equal amounts of all samples in the experiment is labeled with Cy2. All samples are then applied on the same gel. The pooled sample acts as an internal standard for every protein spot on each of the gels, and is then used for normalization of all spots, across all gels. This approach reduces the experimental variation, increasing both the accuracy of quantification and the statistical confidence of protein expression differences.

Advantages
To study differential expression of proteins, there is no need to run multiple gels. Loss of proteins even in the low molecular weight range is reduced since no post-electrophoretic processing (fixation or destaining) is necessary. This method is more quantitative than the standard colorimetric staining methods, both with regard to sensitivity as well as linearity.

license: Creative Commons - Attribution Share-Alike - https://creativecommons.org/licenses/by-sa/4.0/


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Proteomics/Protein Separations- Electrophoresis/QPNC-PAGE

Theory
Quantitative preparative native continuous polyacrylamide gel electrophoresis (QPNC-PAGE) is an electrophoresis technique to isolate native or active metalloproteins in biological samples and to resolve properly and improperly folded metal cofactor-containing proteins in complex protein mixtures. Some examples of metalloproteins are metal chaperones, prions, metal transport proteins, amyloids, metalloenzymes, and metallopeptides.

Gel and Buffer Characteristics
The gel is polymerized for a time period of sixty-nine hours at room temperature. That results in a gel which is mechanically stable, homogeneous, and free of monomers or radicals, and therefore, only negligible interactions of the gel with the biomolecules occur. The metalloproteins are not dissociated into metal cofactors and apoproteins during electrophoresis, and purified metalloproteins are isolated in quantitative amounts in different PAGE fractions. Due to the specific characteristics of the gel, the conformation or active structures of the isolated proteins are not changed by this method.

The chemical stability of a specific metalloprotein can be investigated and confirmed by using size-exclusion chromatography.

The buffer used for electrophoresis contains 20 mM Tris-HCl, 1 mM NaN3 and has a pH 10. Due to the specific properties of the gel and buffer solution, most proteins in the sample are charged negatively and migrate from the cathode to the anode in the electric field. The proteins are eluted continuously by a physiological buffer solution and isolated in different fractions.

Analysis and Applications
Metal cofactors eluted in different PAGE fractions can be identified and quantified by mass spectrometry. Since the pure metalloproteins are separated under non-denaturing conditions, the structures of these proteins can be obtained by solution NMR spectroscopy.

Improper folding of metalloproteins results in degenerative conditions like Alzheimer's disease. The quantitative analysis of metallochaperone proteins in biofluids helps in the clinical investigations concerning the structure-function relationships of biologically-active metal cofactor-containing chaperones in those protein-misfolding diseases. QPNC-PAGE combined with biological mass and NMR spectrometries provides valuable information about the metabolisms of important metal cofactors in biological systems.


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Proteomics/Protein Separations- Electrophoresis/IsoElectric Focusing

Isoelectric Focusing(IEF)
In Isoelectric focusing, proteins are separated by electrophoresis in a pH gradient based on their isoelectric point(pl). A pH gradient is generated in the gel and an electric potential is applied across the gel. At all pHs other than their isoelectric point, proteins will be charged. If they are positively charged, they will move towards the more negative end of the gel and if they are negatively charged they will move towards the more positive end of the gel. At its isoelectric point, since the protein molecule carry no net charge it accumulates or focuses into a sharp band.

Immobilized pH Gradeint (IPG) and IEF run
Immobilized pH gradients are used for IEF because the fixed pH gradients remain stable over extended run times at very high voltages. The pH gradients of IPGs are generated by means of buffering compounds that are covalently bound into polyacrylamide gels. IPGs are cast strips with plastic backing sheets and are commercially available in different pH ranges and lengths. They offer high resolution, great reproducibility, and allow high protein loads. Isoelectric focusing is run in the same solutions that are used to extract or solubalize the proteins. The IPG strips with the protein sample must be rehydrated in the rehydration/sample buffer during which protein samples are loaded into the strips. Rehydration can be active or passive. To load larger proteins active rehydration in small voltage is applied.As the sample is electrophorised, the proteins will migrate toward either the anode or cathode, depending on charge. The proteins will stop when they reach their respective pI. After the run in IEF cell, the proteins focus as bands on the strip according to their isoelectric points. The focused strips can be frozen for storage.


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Proteomics/Protein Separations- Electrophoresis/One Dimentional Gel Electrophoresis

 * 1) Gel Electrophoresis
 * 2) One Dimentional Gel Electrophoresis
 * 3) Two Dimensional Polyacrylamide Gel Electrophoresis(2D-PAGE)
 * 4) Differential in Gel Electrophoresis(DIGE)
 * 5) QPNC-PAGE
 * 6) Capillary Electrophoresis
 * 7) Timelines of Electrophoresis
 * 8) Databases
 * 9) Web Pages
 * 10) Online Applications
 * 11) Further Readings

Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE)
This is a very common method of gel electrophoresis for separating proteins by mass. The most commonly used system is also called the Laemmli method after U.K. Laemmli, who was the first to publish a paper employing SDS-PAGE in a scientific study.The proteins are dissolved in sodium dodecyl sulfate (SDS),a detergent that breaks up the interactions between proteins,and then electrophorised. The smallest molecules move through the gel faster, while larger molecules take longer and result in bands closer to the top of the gel.

Gel
The gel used for SDS-PAGE is made out of acrylamide which form crooss-linked polymers of polyacrylamide. Standard gels are typically composed of two layers, one top-most layer called the stacking gel and a lower layer called separating or resolving gel.The stacking layer contains a low percentage of acylamide and has low pH, while the acrylamide concentration of the separating gel varies according to the samples to be run and has higher pH.The difference in pH and acrylamide concentration at the stacking and separating gel provides better resolution and sharper bands in the separating gel.

Procedure
The samples are treated with SDS (sodium dodecyl sulfate),an anionic detergent which denatures the protein by breaking the disulfide bonds and gives negative charge to each protein in proportion to its mass.Without SDS,different proteins with similar molecular weights would migrate differently due to differences in folding,as differences in folding patterns would cause some proteins to better fit through the gel matrix than others.SDS linearizes the proteins so that they may be separated strictly by molecular weight.The SDS binds to the protein in a ratio of approximately 1.4 g SDS per 1.0 g protein [],giving an approximately uniform mass:charge ratio for most proteins,so that the distance of migration through the gel can be assumed to be directly related to only the size of the protein.Proteins may be further treated with reducing agent, such as dithiothreitol(DTT) or TRP(Tributyl phosphine)to break any reformed disulfide bonds and then alkalated with iodoacetamide to prevent reformation of disulfide bonds. A tracking dye like bromophenol blue may be added to the protein solution to track the progress of the protein solution through the gel during the electrophoretic run.

Electrophoresis
The proteins denatured by SDS are applied to one end of a layer of polyacrylamide gel submerged in a buffer.Buffer provide uniform pH and ions for conducting electric potential. When an electric current is applied across the gel,the negatively-charged proteins migrate across the gel to the positive pole. Short proteins will more easily fit through the pores in the gel and move fast, while larger ones will have more difficulty.Due to differential migration based on their size, smaller proteins move farther down the gel, while larger ones stay closer to the point of origin.After a given period of time,proteins might have separated roughly according to their sizes.Proteins of known molecular weight (marker proteins)can be run in a separate lane in the gel to calibrate the gel.

Staining and analysis
Following electrophoresis,the gel may be stained with Coomassie Brilliant Blue or silver stain to visualize the separated proteins.After staining, different proteins will appear as distinct bands within the gel according to their sizes and thus by molecular weights.The molecular weight of a protein in the band can be estimated by comparing it with the marker proteins of known molecular weights.The separated proteins can be cut from the gel and further analyzed by other proteomics techniques.

Isoelectric Focusing(IEF)
In Isoelectric focusing, proteins are separated by electrophoresis in a pH gradient based on their isoelectric point(pl). A pH gradient is generated in the gel and an electric potential is applied across the gel. At all pHs other than their isoelectric point, proteins will be charged. If they are positively charged, they will move towards the more negative end of the gel and if they are negatively charged they will move towards the more positive end of the gel. At its isoelectric point, since the protein molecule carry no net charge it accumulates or focuses into a sharp band.

Immobilized pH Gradeint (IPG) and IEF run
Immobilized pH gradients are used for IEF because the fixed pH gradients remain stable over extended run times at very high voltages. The pH gradients of IPGs are generated by means of buffering compounds that are covalently bound into polyacrylamide gels. IPGs are cast strips with plastic backing sheets and are commercially available in different pH ranges and lengths. They offer high resolution, great reproducibility, and allow high protein loads. Isoelectric focusing is run in the same solutions that are used to extract or solubalize the proteins. The IPG strips with the protein sample must be rehydrated in the rehydration/sample buffer during which protein samples are loaded into the strips. Rehydration can be active or passive. To load larger proteins active rehydration in small voltage is applied.As the sample is electrophorised, the proteins will migrate toward either the anode or cathode, depending on charge. The proteins will stop when they reach their respective pI. After the run in IEF cell, the proteins focus as bands on the strip according to their isoelectric points. The focused strips can be frozen for storage.

Native-PAGE
Native PAGE is used to separate proteins in their native states according to difference in their charge density. Native state of protein means proteins are in properly folded state, not denatured or unfolded state. There are no denaturants present in the gel and buffer in the gel maintains the protein in its native state. Many proteins are shown to be enzymatically active after separation by native PAGE. Thus, it is used for preparation of purified and active proteins. In native PAGE the mobility depends on both the protein's charge and its hydrodynamic size. The charge depend on the amino acid composition of the protein as well as post-translational modifications. The hydrodynamic size and mobility of native protein on the gel will vary with the nature of the conformation. Proteins with compact conformations have higher mobility and larger structures like oligomers have lower mobility. Native PAGE can be carried out near neutral pH to avoid acid or alkaline denaturation to study conformation as well as self-association or aggregation, and the binding of other proteins or compounds. The apparatus is kept cool to minimize denaturation of proteins and proteolysis.


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Proteomics/Protein Separations- Electrophoresis/Timelines of Electrophoresis

 * 1) Gel Electrophoresis
 * 2) One Dimensional Gel Electrophoresis
 * 3) Two Dimensional Polyacrylamide Gel Electrophoresis(2D-PAGE)
 * 4) Differential in Gel Electrophoresis(DIGE)
 * 5) QPNC-PAGE
 * 6) Capillary Electrophoresis
 * 7) Timeline of Electrophoresis
 * 8) Databases
 * 9) Web Pages
 * 10) Online Applications
 * 11) Further Readings

Timeline of Electrophoresis
1930s:  Use of sucrose for gel electrophoresis.

1937:  The official launching of Tiselius' moving boundary electrophoresis (MBE).

1939:  Zone Electrophoresis

1950:  Rich in introducing novel methodologies in zone electrophoresis (ZE)

1950:  Agar Gel electrophoresis

1955:  Introduction of starch gels

1957:  Cellulose acetate Electrophoresis (CAc) by Kohn

1959:  Introduction of acrylamide gels by Raymond and Winstraub

1961:  Electrophoresis in silica gel by Honnegar

1961: IsoElectricFocusing by Svensson

1964:  Electrophoresis in thin layer of sephadex beads by Fasella et.al

1964:  Disc gel electrophoresis Ornstein and Davis

1969:  Introduction of denaturing agents especially SDS separation of protein subunit by Beber and Osborn

1970:  T4 phage proteins were separated using stacking gel and SDS by Laemmli

1975:  2 Dimensional gels: Isoelectric focusing and SDS gel electrophoresis by OFarrell.

1977:  Sequencing gels

1970s:  agars gels

1980s: Immobilized pH gradients

1983:  Pulsed field gel electrophoresis

1983:  Capillary electrophoresis


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Proteomics/Protein Separations- Electrophoresis/Two Dimensional Polyacrylamide Gel Electrophoresis(2D-PAGE)

Theory
Two dimensional polyacrylamide gel electrophoresis (2D-PAGE) is a form of gel electrophoresis in which proteins are separated and identified in two dimensions oriented at right angles to each other.

In this technique, proteins are separated by two different physical properties. The first dimension, isoelectric focusing, separates proteins on the basis of their net charge. The second dimension, SDS-PAGE, further separates the proteins by their mass. Small changes in charge and mass can easily be detected by this method, because it is rare that two different proteins will resolve to the same place in both dimensions.

For further details on gels, staining, and electrophoretic analysis, see the "Gel Electrophoresis" section of this chapter. For further details on the individual dimensions of two dimensional electrophoresis, isoelectric focusing and SDS-PAGE, see the "One Dimensional Electrophoresis" section of this chapter.

Process
The proteins in a focused IPG strip are uncharged (their net charge is 0) because they are at their isoelectric point, so they will not move into the SDS-PAGE gel. To prepare the proteins for movement into the SDS-PAGE gel, the equilibrated strip is treated with detergent SDS. The treated IPG strip is placed on top of the SDS-PAGE gel, submerged in a buffer, and sealed in place with agarose gel. When an electric current is applied across the layered gels, the negatively charged proteins move out of the IPG strip and migrate through the SDS-PAGE gel. Each protein moves differently through the gel matrix depending on its size. The separation of proteins is distributed roughly according to size (molecular weight), like other electrophoresis methods.

Visualization and Analysis
The resolution of 2D-PAGE gels is somewhat different than that of one-dimensional gel electrophoresis. Proteins are rarely resolved as discrete bands; rather, they are resolved as spots cascading down the gel. These gels can be stained in much the same way as other electrophoresis techniques, and adding another dimension of separation does not in any way hinder visualization.

Some analysis of 2D-PAGE gels has a direct complement in one-dimensional electrophoresis; the determination of molecular weight is done the same way in 2D-PAGE as in the simpler method. Analysis of 2D-PAGE gels can become more complicated, however, because it is not as easy to discern the identity of proteins with the naked eye. For careful analysis, image analysis software, such as Delta2D, can be useful. Delta2D can quantify protein spots, match images, compare corresponding spots and intensities of related gels, prepare gel data reports, remove background patterns, and integrate image information to databases. There are also many other software packages available.

Advantages and Disadvantages
Using 2D-PAGE, hundreds to thousands of polypeptides can be analyzed in a single run. The proteins can be separated in pure form from the resultant spots. The spots can be quantified and further analyzed by mass spectrometry, depending on their resolution. Polypeptides can also be probed with antibodies and tested for post-translational modifications. 2D-PAGE is also used to study differential expression of proteins between cell types.

The disadvantages of this technique include a large amount of sample handling, limited reproducibility, and a smaller dynamic range than some other separation methods. It is also not automated for high throughput analysis. Certain proteins are difficult for 2D-PAGE to separate, including those that are in low abundance, acidic, basic, hydrophobic, very large, or very small. Through advancements in this technology, scientists are, however, able to separate low abundance proteins with moderate efficiency.


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User:Carlwev

Here I am

title: User:Carlwev


text:
Digital Signal Processing/Windowing

Most digital signals are infinite, or sufficiently large that the dataset cannot be manipulated as a whole. Sufficiently large signals are also difficult to analyze statistically, because statistical calculations require all points to be available for analysis. In order to avoid these problems, engineers typically analyze small subsets of the total data, through a process called windowing.

Windowing Introduction
Windowing is the process of taking a small subset of a larger dataset, for processing and analysis. A naive approach, the rectangular window, involves simply truncating the dataset before and after the window, while not modifying the contents of the window at all. However, as we will see, this is a poor method of windowing and causes power leakage.

Applying Windows
Application of a window to a dataset will alter the spectral properties of that dataset. In a rectangular window, for instance, all the data points outside the window are truncated and therefore assumed to be zero. The cut-off points at the ends of the sample will introduce high-frequency components.

Consider the system H(z), with input X(z) and output Y(z). We model this as:

 * $$Y(z) = X(z) H(z)$$

If we have a window with transfer function W(z), we can mathematically apply the window to our signal, X(z) as such:

 * $$\hat{X}(z) = X(z) W(z)$$

Then, we can pass our windowed signal into our system, H(z) as usual:

 * $$\hat{Y}(z) = \hat{X}(z) H(z)$$

Leakage
If our signal is a lowpass or passband signal, the application of a window will introduce high-frequency components. Power from the original signal will be diverted from the specified frequency band into the high-frequency areas. This redistribution of power from a target band to the upper frequencies is known as leakage.

If we look at a rectangular window, we know from duality that the frequency response of that window is a sinc function, which has non-zero values all the way out to both positive and negative infinity. Convolution of the sinc function with any narrow-band signal is going to cause a very spread-out spectrum.

Hamming
function of hamming

0.54+0.46cos(2*pi*n/N-1)

Blackman window
0.42+.5cos(2*pi*n/N-1)+.08cos(4*pi*n/N-1)

Greatest stop band attenuation of mentioned windowing techniques at the expense of a larger transition band.

The eq. for the symmetric 4-term Blackman Harris window of length N is....

w(n) = a0 - a1*cos(2*pi*n/N-1) + a2*cos(4*pi*n/N-1) - a3*cos(2*pi*n/N-1); 0 <= n <= N-1

a0 = 0.35875   a1 = 0.48829    a2 = 0.14128   a3 = 0.01168

title: Digital Signal Processing/Windowing


text:
Biblical Studies/New Testament Commentaries/The Gospel of John/Chapter 17

His prayer
Verses 1-26, sums up Jesus teachings, Jesus begins to pray to the Father for different reasons. First, He prays for Himself (verses 1-5) then He continues and begins to pray for His apostles (verses 6-19 and 24-26) and for all believers and the world (verses 20-23). This prayer seems to combine all things done, past present, and future. Each prayer depends on the one before it with Jesus being the foundation. (Petersen).

Jesus' prayer is the culmination of all of his ministry in the world. Jesus prays for God to be glorified in what is about to come through him. Jesus also prays for his disciples and all believers that God would bless them and the Holy Spirit would be revealed to them and be present in the believers' lives.

Verses 1-5: Jesus Prays for Himself
Throughout this Gospel we have been told that his 'hour had not yet come' (the first occasion in John 2:4). Jesus begins to express the desires of His heart in verses 1-5. Now, it 'has come'. Now he prays that he may be glorified. No one can glorify him but the Father and he prays that he may so accept it as to bring glory to his Father in turn. His “hour” had come for the Father to “glorify Thy Son,” (NAS) which would be through His death, resurrection, and ascension. He will be glorifying his Father by doing his will.

In verse 2 Christ was given two things; the “authority over all mankind” and the power to “give eternal life.” (NAS)

He continues (v.3) to define this “eternal life” as knowing “the only true God” and Jesus Christ whom he had sent. This idea of mankind “knowing” God could have several interpretations. Some believe that knowing God was literal and so the verse seemed ambiguous. However, others believe that “knowing” God meant that mankind would no longer need a mediator and could have a personal knowing of God and Jesus Christ.

In verse 4 Jesus talks of His glorification and it should be noted that the language used in this verse implies that this glorification had already been done. “I glorified Thee on the earth, having accomplished the work which Thou hast given Me to do.” (NAS)

In verse 5 Jesus states: "Now, Father, glorify Me together with Yourself, with the glory which I had with You before the world was.” (NAS) Here some believe that Jesus was saying He had finished what he came into the world to do, but in order for Him to come into the world, Jesus had to set aside His glory and become a servant. However, Jesus is now asking for God to reinstate His glory as He left the world. (Philippians 2:5-11) By asking for glory at God's side Jesus is also telling us that he is one with God and the Holy Spirit, thus reaffirming the holy Trinity of Christian belief, a very important concept in the times that were to come. Through this belief, because all three are one in the same they cannot bestow glory on themselves, but have to ask for it from each other.

Verses 6-19: Jesus Prays for His Disciples
This section of John Chapter 17 contains much of the essence of Jesus' prayer. He is specifically praying for those closest to him, the disciples. In his prayer, Jesus details to his heavenly Father what has come to pass, what is, and what will be. However, in all cases, the name of God is glorified. The circumstance is special however; this is because Jesus' motives in the prayer lie with his disciples and their acceptance of God's word (aka Jesus). "...And glory has come to me through them...Holy Father, protect them by the power of your name--the name you gave me--so that they may be one as we are one (John 17:11). Here Jesus asks that the disciples be granted unity. However, he is not just asking for simple agreement; he is asking for the ultimate kind of unity: the unity of the Father, the Son. For those who say Jesus is God because he and the father are one this verse unravels that argument. He prays for the same oneness for his followers as he and the father share. Would that make his followers God? Hardly.

The Message of Christ's Prayer "I pray for them. I am not praying for the world, but for those you have given me, for they are yours" Christ's words in verse 9 when he speaks of the disciples are somewhat unclear, and perhaps troublingly so.

He mentions that he is not praying for "the world. The next passage led me to believe that he was praying for the people within the world, but even then, it was only for the believers. In verse 20 Christ says "I pray also for those who will believe in me through their message." There is no mention of those who will not believe in the message. Are we to assume the worst?

Yes we flippantly tell strangers "Jesus loves you", but what does Jesus say? He says he loves his own, those who love the Father and Jesus and demonstrate it by obeying the commands of the Gospel.

Jesus commands us to love everyone including our enemies but he never says he loves sinners, he says he loves those who remain faithful to the Father and his gospel. His love is conditional. However the father's love is universal. He loves all humans.

This chapter contains the prayer of Jesus to the Father, which is overheard by His disciples. The first few verses directly concern Jesus himself, but the bulk of the prayer is for the apostles and those who would become believers later. One of the amazing things about His prayer is that Jesus did not pray just for the apostles, but for those who would believe through their word. The apostles would preach the word, convert many people to Christ. In addition, they would leave on record by their writings the gospels and the many letters.

One of the great questions of Christianity is that of Jesus' relationship to God, and much of that has centered around John's gospel because of its more advanced theological identity. Many point to passages such as John 10:30 in which Jesus proclaims, "I and the Father are one," to suggest that John saw Jesus and God as being equal. While that may certainly be the case if one reads verses 21-23 that says, "21 that all of them may be one, Father, just as you are in me and I am in you. May they also be in us so that the world may believe that you have sent me. 22 I have given them the glory that you gave me, that they may be one as we are one: 23 I in them and you in me. May they be brought to complete unity," it seems rather clear that Jesus is referring to a oneness of mind and purpose and not a oneness of power or physical being. If the disciples are able to be one in the same sense that Jesus is one with God, it only makes sense if read as being united in purpose and thought, as is alluded to in verse 23.

Jesus' prayer was that all the disciples might be one in him and in the Father. He prayed that all who truly believed might ultimately be with him where he was to behold the glory he would have with God (J. Boulton, 2006).

The Revelatory Work of Jesus In 17:25-26, we have more language of being sent. Jesus says, to his Father, whom he calls "Righteous," "The world does not know you, but I know you; and these know that you have sent me." The disciples now know that Jesus came from the "Righteous God." Yet these verses imply that the disciples are able to know this only because Jesus "made [God's] name known to them." It has been said in earlier chapters that the work of the Paraclete is "the revelation of Jesus himself to his disciples," (Smith 319). Jesus says in 17:26, "...I will make it known, so that the love with which you have loved me may be in them, and I in them."

God loves everyone but has a special love for believers.

Chapter 18
The Route to the Olive Grove of Gethsemane

Then Yahshuah, and the eleven, sang the Passover Hallel hymn which contains these lines Psalm 118 They compass me about like bees; they are quenched as the fire of thorns; verily, in the name of Yah I will cut them off. Thou didst thrust sore at me that I might fall; but Yah helped me. Yah is my strength and song; and He is become my salvation. The voice of rejoicing and salvation is in the tents of the righteous; the right hand of Yah does valiantly. The right hand of Yah is exalted; the right hand of Yah does valiantly. I shall not die, but live, and declare the works of Yah. Yah has chastened me sore; but He has not given me over unto death…I will give thanks unto You, for Thou hast answered me, and art become my salvation. The stone which the builders rejected is become the chief corner-stone. This is Yah 'S doing; it is marvelous in our eyes. …O give thanks unto Yah, for He is good, for His mercy endures forever. (Psa.118:1-29)

After they sang, they went out of Yerushalayim through the Eastern gate, down through the valley of Gey-hinnom, crossed a bridge over the Kidron brook and ravine, and went up the Mount of Olives to an Olive Grove called Gethsemane (the Place of the Olive Press). The valley of Gey-hinnom (Gehenna) is where Solomon defiled and sacrificed hundreds of young girls. The Kidron ravine is the path David took when he was forced to flee Jerusalem by his son Absalom. David went up the Mount of Olives weeping.

Judas Comes With the Rogues to Arrest Yahshuah     Jn.18

They could see torches, and people, moving into the garden coming towards them. It was Judas Ben Shimon of Keriot with a mob sent by the elders and chief priests (Sahmmai and Gamaliel). There were Temple guards with swords, and a band of Pharisees with staffs and clubs, and some rogue Mosaic Law experts. Judas had received the thirty pieces of silver and was about to betray Yahshuah. Yahshuah fully realizing what was happening went to meet them and asked, “Who are you looking for?” “Yahshuah of Netzeret.” “I am he.” As soon as Yahshuah said ‘I am he,” the mob drew back and fell to the ground as if an invisible force had knocked them down. Yahshuah asked them again, “Who are you looking for?” Rising to their feet, they said again, “Yahshuah of Netzeret.” “I told you I am he. If you are only looking for me, then let the other men here go free.” he said this to fulfill the prophecy, Of those that you gave me, I lost none.

Wounds made by a friend are intended to help, but an enemy's kisses are too much to bear. Prov 27:6 And as I watched, I saw Azazel approach Him and he kissed Him on the face and then stood behind Him. And I said, "0 Eternal One! Who is the Man insulted and beaten, who is worshipped by the nations and kissed by Azazel?" And He answered and said, "Hear Abraham! The Man you saw insulted and beaten and yet worshipped by many, He is the 'Relief' granted by the nations to the people who proceed from you, in the last days, in the twelfth hour of the age of ungodliness. But in the twelfth hour of my final age will I set up this Man (the Messiah, Yahshuah) from your generation, whom you saw issue from among my people, and all who follow will become like this Man, and such as are called by me will join the others, even those who will to change within themselves. The Apocalypse of Abraham 1:55-56

Peter Tries to prevent the Arrest 		   Jn. 23

Then Peter drew one of the two swords and struck the High Priest’s servant Melekh, and cut off his right ear. Yahshuah restrained Peter and asked, “Shall I not drink the cup that Father has prepared me for? Put up your sword, all who live by the sword will die by the sword. Do you not understand that I could call 60,000 angels and Father would send them to me?” The chariots of Yah are ten thousands and thousands upon thousands. From the Dead Sea Scrolls Psalm 68:17 And Elisha began to pray and said: O Yah, open his eyes, please, that he may see. Immediately Yah opened the attendant's eyes; so that he saw; and, look! the mountainous region was full of horses and war chariots of fire round about Elisha. 2Ki.6:17 Wisdom is better than weapons of war. Ecclesiastes 9 :18 Anyone shedding man's blood, by man will his own blood be shed, for in God's image he made man. Ge.9:6 Yahshuah went to the wounded servant and touched the wound, and the bleeding stopped, and instantly his ear was back on his head as good as new. Then he said to the mob and the soldiers, “Am I a wild bandit that you have to come after me with swords and clubs in the dark of night to arrest me? Do you think I am leading a gang of assassins and zealots? I am a teacher, and I have been peacefully sitting in the Temple every day this week in broad daylight telling people about the Kingdom of Yah. Apparently, no one thought I was a wild bandit then. So who has changed your mind about me? I will tell you who it was, it was Azazel, the devil, for he always does his work in the dark, the Prince of the Power of Darkness reigns over you, and so you do his bidding.” Then they seized him and led him away. All the Apostles fled. A soldier grabbed one disciple by his robe, but he wiggled out of it, and fled away naked.

The Ransom was paid in the garden By allowing the forces of darkness to seize him Yahshuah paid the ransom. The ransom was paid at that moment in the garden. A sinless man surrendered himself as a prisoner exchange to Azazel. If he can remain sinless until death, he will break Azazel’s power to keep humans forever in the land of death. Azazel is confident that with a little torture Yahshuah will be easy to break. Azazel is unaware that if Yahshuah dies sinless, the curse of the Torah will fall upon him. “Cursed is he who accepts a payment and then slays an innocent man.” (Deut. 27:25)

Yahshuah in the House of Ananus, the Ringleader of the Rogues

The Temple guards first took him to the house of Ananus, the retired illegal High Priest; he was the ringleader of the rogues who had appointed his son-in-law Kayafa as his illegal successor.

222     Peter Denies Knowing Yahshuah Two Times

When the Apostles fled, two of them, Peter and Yahchanan had followed the mob with Yahshuah. Yahchanan had connections in the High Priest’s house and easily entered the courtyard. After a while, he spoke to the woman gatekeeper and arranged for Peter to enter. As Peter came in she asked, “Aren’t you one of Yahshuah’s followers? “No, I am not,” he said. The Temple guards had built a fire in the courtyard and many slaves and other people were sitting around it warming themselves. Peter came and began to warm himself. One of the slave girls sitting there recognized him and said, “There is one of his followers. I saw him one day with Yahshuah.” Peter denied it saying, “By my oath, I do not even know him miss, you are mistaken.” “I am sure I saw him with Yahshuah of Netzeret,” she said, Peter heard a rooster crow off in the distance.

Ananus ’s Guard Strikes Yahshuah

Inside Ananus’ house, Ananus questioned Yahshuah about the Gospel of the Kingdom and about his followers. Yahshuah said, “My teachings were never hidden. I said everything in public in the synagogues and in the Temple. Why do you question me? Ask the people who heard me speak, they will tell you what I taught.” One of the guards struck Yahshuah in the mouth saying, “How dare you talk to the High Priest like this.” Yahshuah, answered, “You know he has not been the High Priest for many years, and when he was in office, he was not legally the High Priest, but a pretender. Therefore if I have said anything evil what was it? And if I broke no law, then why did you hit me?” “And the men that held Yahshuah mocked him, and smote him. And when they had blindfolded him, they struck him on the face, and asked him, saying, Prophesy, who is it that smote you? And many other things blasphemously spake they against him”. (Luke 22:63-65)

Peter Denies Yahshuah the Third Time

Peter moved away to the porch of Kayafa’s house. One of the high Priest’s slaves saw him and said. “He is one of them. I saw him in the garden near my uncle Melekh.” Peter said, “I swear I do not know him.” The slave said, “Listen to the way he speaks, he is a Galilean.” Peter said, “On my oath, may curses fall upon me if I am not telling the truth.” Immediately he heard a rooster crow the second time and he remembered what Yahshuah had said. Meanwhile Ananus had ordered Yahshuah transferred across the courtyard to the house of Kayafa. Yahshuah came onto the porch and he and Peter looked at each other. Peter ran from the courtyard and into the city weeping bitterly. Roosters only crow at dawn, but the Romans soldiers used to blow an instrument 4 times during the night that sounded like a rooster. It would now be about midnight.

Yahshuah Tells the Rogues He is the Messiah

When it was morning Kayafa convened an illegal trial the elders of the people and the chief priests and the scribes came together, and led him into their council, saying, Are you the Messiah? tell us. And he said unto them, If I tell you, you will not believe me: And if I also ask you a question, you will not answer me, nor let me go. (Luk 22:67-68) Kayafa brought in false witnesses to condemned Yahshuah. The witnesses however kept contradicting each other. At last, two witnesses came in and said, “This man said if we tear down the Temple made with hands, in three days he could build it with his own two hands.” “With one hand,” one of the witnesses said. “No with two,” the other said. The two witnesses began to argue and Kayafa said to Yahshuah, “Well what you say was it with one hand or two?” Moses wrote in the Scroll of Deuteronomy: On the evidence of two or three witnesses the death sentence shall be executed; a person must not be put to death on the evidence of only one witness. The hands of the witnesses shall be the first raised against the person to execute the death penalty, and afterwards the hands of all the people. So you shall purge the evil from your midst. Deut 17:6-7 False witnesses have risen up against me and they breathe out violence. From the Dead Sea Scrolls Psalm 27:12 Wicked and deceitful people have opened their mouths against me and have spoken against me with lying tongues. They surround me with words of hatred and attack me without a cause. From the Dead Sea Scrolls Psalm 109:2

Yahshuah was silent. Kayafa asked, “Have you nothing to say?” Yahshuah said nothing.” He was hard pressed and he was letting himself be afflicted; yet he would not open his mouth. He was brought like just like a sheep to the slaughter; and like a ewe that before her shearers has become mute, he also would not open his mouth. Is.53:7 Kayafa stood up and shouted, “I want you to swear by the living Eloah and tell us whether or not you are the Messiah the son of Yah.” Yahshuah said, “I am the Messiah, the son of Yah, and furthermore this mortal man will ascend to heaven, and you will see him seated at the right hand of Yah riding on the Shokeyn cloud of Glory.” It is written in the Scroll of Daniel: I kept on looking in the visions of the night and, see there! with the clouds of the heavens someone like a mortal happened to be coming ; and to the Ancient of days he gained access, and they brought him up close even before that One. And to him there was given him, a rulership and dignity and a kingdom, that all peoples, national groups, and languages should all serve him. His rulership is an everlasting rulership that will not pass away, and his kingdom one that will not be brought to ruin. Dan.7:13 Then Kayafa broke the law and tore his own robe Subsequently Moses said to Aaron, and to Eleazar and to Ithamar, his sons: Do not let YOUR heads go ungroomed, and YOU must not tear YOUR garments, that YOU may not die. Lv.10:6

Then Kayafa screamed, “He has blasphemed. You heard his blasphemy. We need no more witnesses. He is guilty of death.” It is written: So the abuser of Yah’s name should be put to death without fail. The entire congregation should without fail pelt him with stones. Lv.24:16

Then everyone, including the slaves, rushed at him and began spitting on him and hitting him. Someone put a blindfold on him and hit him saying, “Prophesy O great Adoni and tell us who hit you.”

Judas Returns the Thirty Pieces of Silver and Commits Suicide

After they had sent Yahshuah to the dungeon, Judas Ben Shimon of Keriot came in and said to the chief priests (Gamaliel and Shammai) and senators, “I have betrayed an innocent man.” They said, “That is your affair, we had nothing to do with it.” Judas went to the Temple and hurled the thirty pieces of silver into the treasury. Then he hung himself in a tree. As he was hanging, the limb broke and he fell to the ground. His stomach burst open and his entrails spilled out. Therefore, the Jews called it the haunted place, Aceldama, the Field of Blood. But you, O God, will bring them down into the pit of destruction; bloody and deceitful men will not live out half their days; but, I will trust in you. Ps.55:23

The Thirty Pieces of Silver Used to Buy a Cemetery for the Poor

When the priests found the coins, the next day they said, “This is blood money, we cannot put it in the treasury, and so they used it to buy a cemetery for paupers on the site of an old pottery field and thus was fulfilled the Prophecies. Cast it into the treasury, the goodly price that I was prized by them. And I took the thirty pieces of silver and cast them into the treasury, in the house of Yah...and gave them for the Potter’s field. -Zech. 13

The Rogues Have Their First Meeting With Pilate Asking Him to Execute Yahshuah

At dawn, the rogue chief priests (Gamaliel and Shammai), rogue Mosaic Law Experts, and rogue Senators met and decided to take Yahshuah to the Roman Prefect, Pilate. They took him to the government headquarters, the Praetorian. The kings of the earth take their stand and high officials themselves have massed together as one against Yah and against his anointed one. Ps.2:2

If Jews enter a non-Jewish dwelling, they are considered defiled, and none of these rogues wanted to be defiled, or they would not be able to eat the coveted Passover meal, so they requested Pilate to come out to them. Pilate is believed to have come from Scotland, he ruled from 26 to 36 AD. The Jewish Encyclopedia says, “His administration was characterized by corruption, violence, robberies, ill treatment of the people, and continuous executions without even the form of a trial…Pilate looted “funds from the sacred treasury in order to provide for the construction of an aqueduct”…when the citizens protested he sent among the crowds disguised soldiers carrying concealed daggers, who massacred a great number, not only of the rioters, but of casual spectators…Pilate's last deed of cruelty, and the one which brought about his downfall, was the massacre of a number of Samaritans who had assembled on Mount Gerizim to dig for some sacred vessels.” http://www.jewishencyclopedia.com/view.jsp?artid=312&letter=P&search=pilate When Pilate saw them with a prisoner, for Yahshuah was bound with a chain, he asked, “What are the charges?” They tried to dispense with the charges saying, “If he wasn’t a terrible criminal we would not have bothered you so early in the morning.” Pilate said, “Why don’t you try him in your own law court?” They said, “As you know since you Romans took over, you took away our right to execute criminals.” This was to fulfill the prophecy that he would be lifted up and hung in a tree, for that is how the Romans executed people. (The Jews stoned, burned or beheaded them).

Here are the charges: •	SEDITION

•	TAX EVASION

•	IMPERSONATING A KING

•	IMPERSONATING THE MESSIAH

•	IMPERSONATING GOD

“This man is a wizard, shaman, warlock, and an insurrectionist. He is stirring up the people telling them not to pay taxes to Caesar, and claiming to be the Messiah, the King of the Jews.” Pilate said to Yahshuah, “What do you have to say about all these charges?” Yahshuah said nothing and Pilate marveled.

Yahshuah is Questioned by Pilate

Pilate ordered Yahshuah to be brought inside to the Judgment Hall, and then asked him, “Are you the King of the Jews?” Yahshuah asked, “Are you truly trying to understand this for yourself? Or are you only trying to confirm what the illegal chief priests (Gamaliel and Shammai) said about me?” Pilate said, “Only a Jew would care about these things, do I look like a Jew? Your own nation and chief priests, Shammai and Gamaliel, have brought you to me. What did you do?” Yahshuah denies being any part of the Jewish/Israeli nation: Yahshuah said, “My Kingdom (my nation) is not of this Cosmos. If it were, my followers would have fought to keep me from being arrested.” Pilate said, “Ah ha! So you are a King then?” Yahshuah said, “Yes I am. But my kingship is not to reign over others as earthly kings reign, but to deliver the truth. Everyone who is on the side of truth will listen to me. a good ear will hear wisdom with all desire. Ecclesiasticus (from the Latin Vulgate): 3:31

Pilate turned his back, and as he walked away, he rhetorically quipped, “What is the truth?” (It is clear by Pilates reply that he, like most of the elite in Rome, and followed the philosophy Pyrrho, the Skeptic, which denied that truth can be known. They believed only fools spoke of the truth.)

Pilate Tells the Rogues to Take Yahshuah to Antipas

Pilate went out to the priests and senators and said, “He is innocent of any crimes. We Romans do not kill a man unless he commits a serious crime. It is not a crime to say you are a king of another Cosmos come to deliver the truth.” The rogues began to accuse Yahshuah more fiercely saying, “This man has been preaching rebellion throughout the nation. He began in the Galil region and now he is here”. When Pilate detected that Yahshuah was from the Galil region, he said. “King Antipas is in town for the feast. If this man is a Galilean, take him to Antipas.”

Yahshuah Before Antipas

So the angry rogues took Yahshuah to Antipas. Antipas was fearful and delighted. He feared Yahshuah might be John, the Baptizer, come back from the dead, and he was delighted for he had heard much about Yahshuah and wanted to meet him, hoping to be entertained by a miracle. Antipas asked him many questions, but Yahshuah stood in complete silence. The rogues stood shouting out the charges against him, but he said not a word. He was oppressed, and he was afflicted, yet he opened not his mouth: he is brought as a lamb to the slaughter, and as a sheep before her shearers is dumb, so he opened not his mouth. Isa 53:7

It is written in the Scroll of Jeremiah: But I was like a gentle lamb led to the slaughter. And I did not know it was against me that they devised schemes, saying, ‘Let us destroy the tree with its fruit, let us cut him off from the land of the living, so that his name will no longer be remembered!’ From the Dead Sea Scrolls Jerm 11:19 Antipas and his guards began to mock and ridicule him and treat him despicably. They brought some bright white cloth interwoven with silver specks that dazzled in the sun. They fashioned a king’s costume for him then sent him back to Pilate. From that day forward, Antipas and Pilate, who had been enemies, became fast friends.


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Wikijunior talk:Human Body/Heart

Should "heart" be capitalized? I don't think it's a proper noun. Elocina 00:16, 6 April 2007 (UTC)

You're right. It shouldn't be capitalized. -- xixtas talk 00:36, 6 April 2007 (UTC)

title: Wikijunior talk:Human Body/Heart


text:
Proteomics/Protein Separations- Electrophoresis/Timeline of Electrophoresis

 * 1) Introduction to Electrophoresis
 * 2) Gel Electrophoresis
 * 3) One Dimentional Gel Electrophoresis
 * 4) Two Dimensional Polyacrylamide Gel Electrophoresis(2D-PAGE)
 * 5) Differential in Gel Electrophoresis(DIGE)
 * 6) QPNC-PAGE
 * 7) Capillary Electrophoresis
 * 8) Timeline of Electrophoresis
 * 9) Databases
 * 10) Web Pages
 * 11) Online Applications
 * 12) Further Readings

Timeline of Electrophoresis
1930s :  Use of sucrose for gel electrophoresis.

1937 :  The official launching of Tiselius' moving boundary electrophoresis (MBE).

1939 :  Zone Electrophoresis

1950 :  Rich in introducing novel methodologies in zone electrophoresis (ZE)

1950 :  Agar Gel electrophoresis

1955 :  Introduction of starch gels

1957 :  Cellulose acetate Electrophoresis(CAc) by Kohn

1959 :  Arrival of Acrylamide gels (Raymond and Winstraub)

1961 :  Electrophoresis in silica gel by Honnegar

1961 : IsoElectricFocusing by Svensson

1964 :  Electrophoresis in thin layer of sephadex beads by Fasella et.al

1964 :  Ornstein and Davis introduced Disc-Gel Electrophoresis.

1969 :  Beber and Osborn introduced denaturing agents i.e. SDS separation of protein subunit

1970 :  Stacking gel and SDS by Laemmli was used to separate T4 phage proteins

1975 :  2-D gels :Isoelectric focussing and SDS gel electrophoresis by OFarrell.

1977 :  Arrival of Sequencing gels

1970s :  Arrival of Agarose gels

1980s : Immobilized pH gradients

1983 :  Pulsed field gel electrophoresis was invented

1983 :  Introduction of Capillary electrophoresis

Next Section Databases

Subcription Based References

 * Righetti PG, Electrophoresis: the march of pennies, the march of dimes; Journal of Chromatography A. 2005 Jun 24;1079(1-2):24-40. Abstract[]


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title: User talk:CCGS~enwikibooks


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Geometry/Chapter 18

Trigonometry
Trigonometry is the branch of mathematics dealing with the measures of the sides and angles of a triangle. Using trigonometric ratios, one can determine the side and angle measures (i.e. "solve the triangle") using minimal information.

What is a right triangle?
A right triangle is any triangle where one angle measures 90º. A right triangle consists of two small sides, across from the smaller angles, and a longer side, across from the right angle. The sides of a right triangle have special names. The two smaller ones are called legs, and the longer side is called the hypotenuse (pronounced hi-POT-uh-noose). Notice that the bottom-left angle is 90 degrees. The sides labelled "opposite" and "adjacent" are the legs, and the names are in reference to the angle "a". These terms will always be used when discussing sides of a right triangle, and will always be given in reference to some angle. Remember, the hypotenuse is always the longest side and is always across from the right angle.

Practice
Let's practice what we've learned. For the triangle below, fill in the blanks. What is the length of the hypotenuse? _________ What is the length of the side opposte angle "a"? _________ What is the length of the side adjacent to angle "a"?_________

More Practice
Rotating a right triangle does not alter the names of the sides. Consider the following triangle: Can you name the hypotenuse, adjacent and opposite sides? (6 feet is incorrect (should be 5 feet), since h^2 = a^2 + b^2 = 9 + 16 = 25, the square root being 5) Even though this right angle has been rotated, the sides are still named with respect to the angle "a". See if you can identify the sides of the triangle below. Remember that everything is from the point of view of angle "a". What is the length of the hypotenuse? ___________feet What is the length of the side opposite angle "a"? ___________feet What is the length of side adjacent to angle "a"? ___________feet (Two of the answers are incorrect. The opposite angle should be 3 feet, and the adjacent angle should be 4 feet)

Trigonometric ratios
The three most common trigonometric ratios are sine (abbreviated "sin"), cosine (abbreviated "cos"), and tangent (abbreviated "tan"). These trigonometric ratios can be determined contingent on the information you have available. If the angle measure is given, entering sin(x), cos(x), or tan(x) in most scientific calculators (where "x" is the angle measure) will yield the sine, cosine, and tangent of the given value. The three other ratios are cosecant (abbreviated "csc"), secant (abbreviated "sec"), and cotangent (abbreviated "cot" or "ctn"), the reciprocal functions of the sine, cosine, and tangent respectively.

Soh Cah Toa
In a right triangle, if you are given at least two of the side measures, you can determine the sine, cosine, or tangent using a method commonly referred to by the mnenomic device Soh Cah Toa. What this refers to is: The terms opposite and adjacent are in relation to the reference angle, which is the angle your calculations are based upon. In Figure 1, since angle A is the reference angle, then the side opposite of that angle is side a. While there are two sides adjacent to angle A, side b is designated as the hypotenuse so side c is the adjacent side. Therefore, in Figure 1: Please note that "sin(A)" is read as "sine of A," "cos(A)" is read as "cosine of A," and "tan(A)" is read as "tangent of A."
 * Soh: sine = opposite / hypotenuse
 * Cah: cosine = adjacent / hypotenuse
 * Toa: tangent = opposite / adjacent
 * sin(A) = a/b
 * cos(A) = c/b
 * tan(A) = a/c

For example, if a = 7 and c = 14, you would need to find the tangent of angle A, as the other two ratios require the hypotenuse which is unavailable. As the tangent is the opposite divided by the adjacent, you would get tan(A) = 7/14, which can be simplified to one-half.

What now?

 * Note: Please make sure first that your calculator gives angle measures in degrees.

When determining the measures of a triangle, the sines, cosines, and tangents have a function: determining the angle measure. Above, it was determined that the tangent of the reference angle, A, is 1/2. If you entered tan-1(1/2) into a scientific calculator, you will see that you will get the angle measure of A, which is approximately 26.6°. You now have two angle measures: 26.6° and 90°, since this is a right triangle. Since the sum of all angles in a triangle is 180°, subtract the two known angle measures from 180 and you will get the third angle measure: 63.4°.

Solving the side and angle measures of triangles will be covered in more depth in Chapter 19.

title: Geometry/Chapter 18


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Knowledge Management Cases in Asia

This e-book is created by forty-one education students at The University of Hong Kong. It contains actual cases of KM application in various Asian organizations, which includes the beverage industry, a law enforcement unit, a public transport corporation, the telecommunication industry, a non-government organization, a school, and a Chinese dining corporation.

In this e-book, the reason for KM implementation in the industries, strategies employed, problems encountered and some suggestions for future development are recorded. Some of the names and facts pertaining to the organizations may have been modified in order to protect the privacy of the people involved.

 * Samuel Kai Wah Chu, Ph.D.
 * Assistant Professor
 * Division of Information & Technology Studies
 * Deputy Director, Centre for Information Technology in Education
 * Faculty of Education, The University of Hong Kong
 * Pokfulam Road, Hong Kong
 * E-mail: samchu@hku.hk
 * Homepage: http://www.edu.hku.hk/samchu/

Table of contents

 * /Implementation of Knowledge Management in Asia's Beverage Industry/
 * /Knowledge Management in a Law Enforcement Unit/
 * /A Case Study on the Effects of Implementing a Customer Knowledge Management System to a Public Transport Corporation/
 * /Knowledge Management Practice in Telecommunication Industry/
 * /Knowledge Management Pioneer of Non Government Organization in Asia/
 * /Implementation of Knowledge Management in Telecommunication Industry/
 * /A case study of how knowledge management be applied in a school environment in Hong Kong/
 * /Knowledge Management of a Chinese Dining Corporation in Hong Kong/

title: Knowledge Management Cases in Asia


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title: User talk:147.8.110.145


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Hacking/Introduction/Conventions

 * Italic
 * Used for file names and directory names. Also used to introduce new terms.

 * Used to indicate commands, command lines, and command line switches.
 * Used to indicate commands, command lines, and command line switches.

 * Used to indicate user-defined input; the user creates the text.
 * Used to indicate user-defined input; the user creates the text.

 * Used in interactive examples to indicate literal user input.
 * Used in interactive examples to indicate literal user input.

 * Preformatted text blocks
 * Used for code blocks and multi-line shell output examples where whitespace matters

 * [ ]
 * Brackets surround optional inputs; omit the brackets when supplying these inputs.

title: Hacking/Introduction/Conventions


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How to Use Rhino Mocks/Testing Events

Rhino Mocks can be used to test events on interfaces. For example, let's assume an interface that represents an editor view. This might in turn be implemented by an untested, lightweight user control in a WinForms application: We'll be testing the presenter class of this view. An NUnit test that asserts some action takes place due to this event being raised might look like this:

title: How to Use Rhino Mocks/Testing Events


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Instructional Technology/Instructional Design is science, philosophy or technology?

What is design, technology, and philosophy?
Before the discussion of science, philosophy, and technology, there should be comparison and explanation of these concepts. It is better to take a look at these pages:

 * What is Design?
 * What is Technology?
 * Technology is Related to Science?
 * What is philosophy?

Instructional Design is science?
Rowland (1993) explains that both Charles M. Reigeluth and Rita Richey emphasize the science of instructional design because "they feel that design principles accurately predict future phenomena - in this case, learning - and therefore can be used to prescribe instructional events" (p.89) Based on this statement, one can say that instructional designers seek understanding and then apply the principles of design while they are engaged in designing instruction; therefore, instructional design is a science.

Apart from being able to predict future phenomena, and prescribe instructional events, the complexities of the design process require scientific principles and approaches to handle instructional problems. For example, instructional design employs empirical methods in analysis, design, and repeated tryout and revisions to validate the effectiveness of the instructional materials. Through experiments, science simulates natural processes in controlled environments in order to better understand natural phenomena. If through the creation and design of instruction, learners can exhibit observable changes in behavior, knowledge and skills which can be attributed to the use of the instruction (or such attributes can be predicted in a defined instructional context), then why can't instructional design be categorized as science?

In a multifaceted definition of instructional design as a process, a discipline and a reality, Penn State staff also view instructional design as "the science of creating detailed specifications for the development, implementation, evaluation, and maintenance of situations that facilitate the learning of both large and small units of subject matter at all levels of complexity." (Adapted from "Training and Instructional Design", Applied Research Laboratory, Penn State University )

Instructional Design (ID) is philosophy?
Whether you are a practicing instructional designer or a student learning in the field of Instructional Design, you will make instructional and learning decisions for others and yourself. Your design decisions are based on your own beliefs and values. Smith and Ragan (2005) state "fields of study, such as instructional design, do not have educational philosophies; people who study in these fields do" (p.18). In other words, instructional designers bring to their craft their own unique ideas, beliefs and values about the process of education and this serves as a backdrop which exerts both overt and subtle influence on how designers practice. These practice decisions include how designers choose particular design models, how designers carry out assessments, what evaluation measures are chosen, what direction to take when confronted with conficting development needs, just to name a few. Solomon (2000)effectively synthesizes a rich body of literatures in the field and aims to "provide insight into understanding that various ways in which philosophy shapes instructional design practice" (p.23).

There are four critical philosophies had impact on ID theory: (a) modernism, (b)critical theory, (c)pragmatism, and (d) post modernism. They have shaped paradigms that construct ID models. In fact, the ID theory is stemmed from philosophical roots but not directly. The paradigms play critical role here.

In spite of Smith and Ragan's (2005) assertion, Gagne & Merrill (1990); Merrill (1993); and Spector (1994) have explored extensively the philosophy of instructional design. The nature and elements of the building blocks of instructional design, as well as practical application of learning situations have been discussed. Instructional design is based, in part, on the concepts of sequencing instructions, mastery of learning, and is performance-oriented. ID relies on the principles of andragogy, which emphasize experiential learning and other fundamental theories of learning such as cognitivism, behaviorism, and constructivism. According to Spector (2000)learning essentially involves a change in abilities, attitudes, beliefs, capabilities, knowledge, mental models, patterns of interaction or skills. Differentiating and exploring these concepts will further expand the basis of instructional design philosophy.

The advent of distance education added another layer of complexity and Ely (1999) predicts that the growing application, need and interest of distance education may foment the "philosophy of humanistic technology" in the field of instructional technology.

Instruction Design is technology?
In his discussion on the philosophy of instructional technology, Ely says "it may seem strange that in the above discussion of the relationship between a field and a discipline that technology was not considered to be one of the roots of the field. After all, it is the most frequently mentioned term used in the various attempts to give identity to the field" (p.3)

To further muddy the waters of this discussion, we must make mention of the fact that there is considerable thought and numerous writings devoted to the "philosphy of technology." David Blacker of Illinois State University, for example, as written an essay on "The Philosphy of Technology and Education: An Invitation to Inquiry".


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English in Use/Verb Usage

A verb is a part of speech that usually denotes action ("bring", "read"), occurrence ("to decompose" (itself), "to glitter"), or a state of being ("exist", "live", "soak", "stand"). Depending on the language, a verb may vary in form according to many factors, possibly including its tense, aspect, mood and voice. It may also agree with the person, gender, and/or number of some of its arguments (what we usually call subject, object, etc.).

Verbs can be conveyed in many ways. Lets look in particular at the word "wear"

She wears a pink dress - This sentence is made up of three main parts, she, being the noun. Wears, being an action or 'doing' word. Pink, describing the noun about to be added to the sentence and, Dress, the noun which pulls all these words together (giving them all one key point) to complete the sentence.

title: English in Use/Verb Usage