Polyacrylamide Gel Electrophoresis (PAGE)

Hello everyone,

In the following section You can find some important point of Polyacrylamide Gel Electrophoresis

It is most widely used method for analyzing protein mixtures qualitatively. It is particularly useful for monitoring protein purification. As this method is based on separation of proteins according to the size it can also be used to determine the relative molecular mass of proteins. Crossed linked polyacrylamide gels are formed from the polymerization of acrylamide monomer in the presence of smaller amount of N,N methylene –bisacrylamide .

Bisacrylamide is essentially two acrylamide molecules linked by methylene group.It is used as cross linking agent.

Acrylamide monomer is polymerised in a head to tail fasion into long chains and occasionally a bis-acrylamide molecule is built into the growing chain.Thus introducing a second site for chain extension.Polymerization of acryamide  is eg. Of free radical catalysis.

FREE RADICAL CATALYSIS-

Addition of ammonium persulphte and TEMED (N,N,N’,N’-Tetramethylene diamine

S2o8 2- +e-     --------------     So4 -2 +So4-*

Free radical catalysis-free radicals are highly reactive species due to presence of unpaired electron that need to be pair with another electron to stabilize the molecule.

For eg. If free radical presented as R* and M as an acrylamide molecule, then the polymerization can be represented as follows:

R* + M ------------- RM*

RM* + M - ------------ RMM*

RMM* + M ---------------- RMMM*

Care must be used when creating this type of gel, as acrylamide is a potent neurotoxin in its liquid and powdered form.

TEMED-(N,N,N’,N’-tetra methylene diamine)-It catalyses the decomposition of persulphate ion to give free radical .( C6H16N2; MW: 116.21).TEMED stabilizes free radicals and improves polymerization. The rate of polymerization and the properties of the resulting gel depend on the concentrations of free radicals. Increasing the amount of free radicals results in a decrease in the average polymer chain length, an increase in gel turbidity and a decrease in gel elasticity. 

Decreasing the amount shows the reverse effect. The lowest catalytic concentrations that will allow polymerization in a reasonable period of time should be used. APS and TEMED are typically used at approximately equimolar concentrations in the range of 1 to 10 mM

Photopolymerisation

Photopolymerisation is an alternative method that can used to polymerize acrylamide gels.Use of riboflavin. Photodecomposition of riboflavin generates a free radical. 

With love 

-Dixy