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SDS-PAGE (sodium dodecyl sulfate–polyacrylamide gel electrophoresis) is a discontinuous electrophoretic system developed by Ulrich K. Laemmli which is commonly used as a method to separate proteins with molecular masses between 5 and 250 kDa.
Picture of an SDS-PAGE. The molecular markers (ladder) are in the left lane. Polyacrylamide gel electrophoresis (PAGE) is a technique widely used in biochemistry, forensic chemistry, genetics, molecular biology and biotechnology to separate biological macromolecules, usually proteins or nucleic acids, according to their electrophoretic mobility.
C1QBP is 282 amino acid in length and has three homologous subunit with its N-terminal 73 amino acid residues cleaved off to produce mature C1QBP. C1QBP appears as a monomer around 33 kDa on SDS-PAGE gel under both reducing and nonreducing condition but migrates as a trimer on size-exclusion chromatography (gel filtration).
Samples could also be separated first under nonreducing conditions using SDS-PAGE, and under reducing conditions in the second dimension, which breaks apart disulfide bonds that hold subunits together. SDS-PAGE might also be coupled with urea-PAGE for a 2-dimensional gel.
Proteins separated by SDS-PAGE, Coomassie brilliant blue staining. Protein electrophoresis is a method for analysing the proteins in a fluid or an extract. The electrophoresis may be performed with a small volume of sample in a number of alternative ways with or without a supporting medium, namely agarose or polyacrylamide.
SDS-PAGE autoradiography – The indicated proteins are present in different concentrations in the two samples. Proteins , unlike nucleic acids, can have varying charges and complex shapes, therefore they may not migrate into the polyacrylamide gel at similar rates, or all when placing a negative to positive EMF on the sample.
Samples are prepared in a standard, non-reducing loading buffer for SDS-PAGE. No reducing agent or boiling are necessary since these would interfere with refolding of the enzyme. A suitable substrate (e.g. gelatin or casein for protease detection) is embedded in the resolving gel during preparation of the acrylamide gel.
The samples are standardized for protein concentration and then loaded into a polyacrylamide gel for SDS-PAGE. After separation of proteins by molecular weight is complete, the gel is incubated in a renaturing buffer to restore enzymatic activity. During loading, a non-reducing buffer was used to preserve protein disulfide bonds.