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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. Electrophoretic mobility is a function of the length, conformation, and ...
Polyacrylamide (abbreviated as PAM or pAAM) is a polymer with the formula (-CH 2 CHCONH 2-). It has a linear-chain structure. PAM is highly water-absorbent, forming a soft gel when hydrated. In 2008, an estimated 750,000,000 kg were produced, mainly for water treatment and the paper and mineral industries. [1]
Up to 3% gel concentration can be used for separating very tiny fragments but a vertical polyacrylamide gel would be more appropriate for resolving small fragments. High concentrations gel, however, requires longer run times (sometimes days) and high percentage gels are often brittle and may not set evenly.
Proteins of the erythrocyte membrane separated by SDS-PAGE according to their molecular masses. 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.
For a complete protein unstacking the polyacrylamide-gel concentration must exceed 16% T. The two-gel system of "Laemmli" is a simple gradient gel. The pH discontinuity of the buffers is of no significance for the separation quality, and a "stacking-gel" with a different pH is not needed. [15]
In terms of percentage, gels used for protein electrophoresis can be broken down into single-percentage gels and gradient gels. [18] Single-percentage gels are also referred to as linear gels. [20] For linear gels, the selected percentage usually falls between 7.5% and 20%. [18] Common percentage ranges for gradient gels are 4-15% and 10-20%.
"Most agarose gels are made with between 0.7% (good separation or resolution of large 5–10kb DNA fragments) and 2% (good resolution for small 0.2–1kb fragments) agarose dissolved in electrophoresis buffer. Up to 3% can be used for separating very tiny fragments but a vertical polyacrylamide gel is more appropriate in this case.
Both solutions contain acrylamide monomers and catalysts. During polymerization, the acrylamide portion of the buffers co polymerize with the acrylamide and bisacrylamide monomers to form a polyacrylamide gel. These polymerised gels are backed with plastic based backing that allow ease in handling and improve IPG's performance.