One approach is to measure the effect of nicking the DNA. Unconstrained supercoils are released by nicking, whereas constrained supercoils are unaffected. Nicking releases -50% of the overall supercoiling.

Another approach uses the crosslinking reagent psoralen, which binds more readily to DNA when it is under torsional tension. The reaction of psoralen with E. coli DNA in vivo corresponds to an average density of 1 negative super helical turn/200 bp (sigma = -0.05).

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#Unit 13. Methods in Biology

Photopolymerization is an alternative method that can be used to polymerize acrylamide gels. Ammonium persulfate and TEMED are replaced by riboflavin and when the gel is poured it is placed in front of a bright light for 2–3 h.

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#Unit 13. Methods in Biology

Photodecomposition of riboflavin generates a free radical that initiates polymerization.

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#Unit 13. Methods in Biology

Acrylamide gels can be made with a content of between 3% and 30% acrylamide Low concentration are used to where free movement of the proteins is required without any noticeable frictional effect such as flat-bed isoelectric focusing or the stacking gel system of an SDS–polyacrylamide gel.

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#Unit 13. Methods in Biology

Low-percentage acrylamide gels are also used to separate DNA. Gels of between 10 and 20% acrylamide are used in techniques such as SDS–gel electrophoresis, where the smaller pore size now introduces a sieving effect that contributes to the separation of proteins according to their size. 

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#Unit 13. Methods in Biology

Sodium Dodecyl Sulfate (SDS)-Polyacrylamide Gel Electrophoresis is based on the separation of proteins according to size, it can also be used to determine the relative molecular mass of proteins.

#Id: 2490

#Unit 13. Methods in Biology

SDS (H3C-(CH2 )10 –CH2 -OSO3 – Na+ ) is an anionic detergent used to create negative on sample  On average, one SDS molecule binds for every two amino-acid residues.