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Mini-Trans-Blot Electrophoretic Transfer Cell 15

Section 4

Strategies for Optimizing Electrophoretic

Transfer

4.1 Optimizing Protein Transfer

Generally, quantitative elution of denatured high molecular

weight proteins is difﬁcult. The following tactics, alone or in

combination, will increase transfer efﬁciency.

Vary gel composition

Gradient gels are often more effective than single gel

concentrations for elution of a wide range of molecular

weight proteins.

Lower the total monomer to create a more porous gel.

Increase or decrease the percentage of crosslinker. A

5.26% C gel will contain the smallest pore size of all gels

no matter what the concentration of acrylamide. Decrease

in %C will make gels more porous with little loss in

resolution.

grams bis

grams bis + grams acrylamide

Increase transfer time

An initial control should be performed to determine the

time required for complete transfer.

18, 25

Times may vary

from as little as 30 minutes to as long as overnight.

Remember all overnight applications should be performed

at 30 volts to minimize heating problems.

Increase the power

Initial controls should be performed to evaluate the

efﬁciency of increasing the V/cm as well as its effects on

the temperature of transfer. The temperature increase may

change buffer resistance and subsequent power delivered,

as well as the state of protein denaturation, thus affecting

transfer efﬁciency.

%C = x 100

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Bio-rad Mini Trans-Blot® Cell Bedienungsanleitung Seite 21