Question

Explain the purpose of the components of an acrylamide gel.

Answer 1

Polyacrylamide gels are prepared by free radical polymerisation of acrylamide and a crosslinker bis-acrylamide. The polymerisation is initiated by ammonium persulphate with tetramethylenediamine (TEMED) which is used as catalyst.

These gels are used in polyacrylamide gel electrophoresis (PAGE).This technique is useful in separating biological macromolecules, usually proteins or nucleic acids, according to their electrophoretic mobility.

Pore size of gel can be optimized to give the best separation.

Components of acrylamide gel:

(1) Chemical buffer used to stabilize pH to desired value

(2) Acrylamide (C₃H₅NO) when dissolved in water gets polymerised to form long chain polymers.

(3) Bis-acrylamide used as crosslinking agent to form cross links between polyacrylamide chains.

(4) Sodium dodecyl sulfate is a strong denaturating agent used to denature native proteins to unfolded, individual polypeptides. Only used in denaturing protein gels

(5) Urea CO(NH₂)₂ used to increase the entropy of the system by interfering with intramolecular interactions mediated by non-covalent forces such as hydrogen bonds and van der Waals forces.

(6) Ammonium persulphate used for initiation of polymerisation. It provides free radical.

(7) TEMED, stabilizes free radicals and improves polymerization (acts as catalyst)