Isoelectric Focusing

Conventional electrophoresis techniques can separate up to 100 different proteins on one run. Typically, cell or tissue extracts contain thousands of proteins, most of which will not be resolved into single bands using a separation based on any one parameter, such as size or net charge. For any one size range, there is a high…

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Immuno-Electrophoresis / Immuno-Diffusion

Antibodies are produced by the immune system in response to foreign macromolecules. Each antibody binds specifically to one feature (epitope) on one macromolecule (antigen). This allows the use of antibodies for the detection and quantitation of specific proteins in complex mixtures. Antibodies are generally isolated from animal serum, unless they are produced from tissue culture…

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Activity Stains

Samples to be run on native gels should be prepared in a way that minimizes the denaturation of the proteins. Avoid heat, strong detergents, foaming, and over-dilution. In addition, the activity of endogenous proteases must be kept to a minimum. Keeping the sample cold and including protease inhibitors will be helpful in this regard. A…

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Gel Preparation for Native Protein Electrophoresis

The basic protocols for preparing Native PAGE gels are the same as for discontinuous SDS PAGE gels, substituting non-SDS buffers for those containing SDS, as follows: Casting Native Protein Gels Prepare resolving gel and stacking gel casting solutions The table below gives the formulations for native resolving gels from 6 – 12% as well as…

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Sample Preparation for Native Protein Electrophoresis

Samples to be run on native gels should be prepared in a way which minimizes denaturation of the proteins. Avoid heat, strong detergents, foaming and over-dilution. In addition, the activity of endogenous proteases must be kept to a minimum. Keeping the sample cold and including protease inhibitors will be helpful in this regard. A formulation…

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Native Protein Electrophoresis

Proteins run on PAGE in the absence of SDS will separate on the basis of their charge to mass ratio. While native (nondenaturing) PAGE does not provide direct measurement of molecular weight, the technique can provide useful information such as protein charge or subunit composition. Native PAGE also has the potential for separating proteins of…

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Peptide Mapping

Peptide mapping involves controlled cleavage of a pure protein with small amounts of a pure protease to generate peptides of characteristic, reproducible sizes. These peptides can be separated on PAGE to produce a “fingerprint” characteristic of the protein. Peptide mapping can map cleavage sites in an unknown protein, or it can identify an unknown protein…

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Measuring Molecular Weight with SDS-PAGE

The mobility (Rf) of a molecule in gel electrophoresis is determined by its free solution mobility, Y0 (= mobility in a gel of zero %) and the sieving action of the gel matrix. In denaturing protein electrophoresis, the addition of SDS to the electrophoresis buffer uniformly coats the proteins with negative charges, equalizing the charge…

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Casting Gradient Gels

Gradient gels are cast with a higher concentration of acrylamide at the bottom than the top. Gradient gel applications include the determination of protein molecular weights and the separation of molecules which co-migrate on uniform gels. Casting of gradient gels requires a gradient forming apparatus, and is more labor intensive than casting uniform percentage gels.…

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Sample Preparation for SDS-PAGE

SDS is a powerful detergent, which will solubilize many cells and tissues. This greatly facilitates sample preparation for SDS PAGE because most samples will be completely dissolved by heating to 95°C in loading buffer (detailed below). A somewhat stronger loading buffer, containing more SDS and Dithiothreitol (DTT) at a higher pH, can be used for…

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