Bio-Rad Trans-Blot® Cell - Manual (Page 15)

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6. 1.0x TBE (Tris-Borate EDTA) pH 8.3
90 mM Tris-Borate 1 mM EDTA
5x stock solution
54 g Tris base
27.5 boric acid
20 ml 0.5 m EDTA (pH 8.0)
Add 200 ml 5x stock to 800 ml ddH
2
O to make 1.0x working solution.
7. 1x TAE (Tris-Acetate EDTA)
40 mM Tris-Acetate 1 mM EDTA
50-x stock solution
242 g Tris base
57.1 ml glacial acetic acid
100 ml 0.5 m EDTA (pH 8.0)
1x working solution, add 20 ml 50x stock to 980 ml ddH
2
O
Section 4
Strategies for Optimizing Electro-Elution
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. Vary gel composition
a. Gradient gels are often more effective than single gel concentrations for elution of a
wide range of molecular weight proteins.
b. Lower the total monomer to create a more porous gel.
c. 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. An
increase or decrease in %C will make gels more porous with little loss in resolution.
grams bis
grams bis + grams acrylamide
2. Increase transfer time. An initial control should be performed to determine the time
required for complete transfer. 17, 24 Times may vary from as little as 30 minutes to as long
as overnight. Remember all overnight applications should be performed at 30–50 volts to
minimize heating problems. (For long transfers at elevated voltages use the standard
Trans-Blot cell with the super cooling coil.)
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.
4. Reduce buffer strength. Dilution of transfer buffer results in lower current at any given
voltage. This will allow the use of higher voltages without excessive heating.
x 100 %C=