Bio-Rad Trans-Blot® Plus Cell - Manual (Page 23)

TAE (Tris-Acetate EDTA)
40 mM Tris-Acetate 1 mM EDTA
a) Using 50X TAE buffer (catalog #161-0743 for 1L bottles or
catalog # 161-0773 for 5L cube)
20 ml of 50X TAE buffer
980 ml of deionized water (dd H
2
O
b) Using dry reagents (50X stock)
242 g Tris base
57.1 ml glacial acetic acid
100 ml 0.5 M EDTA, pH 8.0
Add 20 ml 50X stock to 980 ml deionized water (dd H
2
O) to make
a 1X working solution.
Section 4
Strategies for Optimizing ElectrophoreticTransfer
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.
1. Improve gel- membrane contact.
Failure of molecules to bind efficiently to the membrane, caused by poor
gel-membrane contact, is often confused with inefficient elution of proteins from
the gel. Poor contact is usually due to excess moisture in the gel-membrane
interface. Use the roller to assure good contact between the gel and membrane.
Proper selection of filter paper thickness will also help assure good compression.
Equilibrate the gel and membrane in transfer buffer for at least 15 minutes prior
to transfer to prevent shrinking of either component during transfer, and to
eliminate reactants such as urea or SDS from the gel.
2. Increase transfer time.
An initial control should be performed to determine the time required for
complete transfer
1,2 . Times may vary from as little as 15–30 minutes to as long
as overnight. Remember all overnight applications should be performed at
10–30 Volts to minimize heating problems.
3. 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.
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