Blotting transfer buffers

The transfer apparatus and the fiber pad used to hold the gel in the gel cassette should be thoroughly cleaned with detergents between use. Dirty fiber pads are a major cause of blot contamination. Also do not re-use transfer buffer, prepare fresh solution... 

Dunn SD. 1986. Effects of the modification of transfer buffer composition and the renaturation of proteins in gels on the recognition of proteins on Western blots by monoclonal antibodies. Anal Biochem 157 144-153. 

Whereas gels are mainly electroblotted immediately after electrophoresis, it is possible to blot Coomassie or Amido Black stained gel too, but with lower efficiency and after soaking in ddH20 for 15 min and an equilibration step in transfer buffer C (see below) for 30 min. 

The actual blotting process may be accomplished by one of two methods passive (or capillary) transfer and electroblotting. In passive transfer, the membrane is placed in direct contact with the polyacrylamide gel and organized in a sandwich-like arrangement consisting of (from bottom to top) filter paper soaked with transfer buffer, gel, membrane, and more filter paper. The sandwich is compressed by a heavy weight. Buffer passes by capillary ac-... 

Prepare transfer buffer (about 4 L for Bio-Rad Trans Blot Cell) and five glass vessels, one of them large enough to accommodate the gel holder... 

If tank blotting equipment is the only option available, then perfectly acceptable results can be obtained. Recommended conditions for tank blotting are overnight transfer (>8 h) at 0.2 A (50 V) with water cooling in Tns/glycme/ 20% methanol transfer buffer (see Section 2.1 ). 

Alkaline blotting39 offers the advantage of efficiency but has been criticized83 as producing blots that hybridize 10 times less efficiently than those transferred in 1 M ammonium acetate, 0.02 N NaOH (after two 15-min washes in 0.25 jVHCI, two 20-min washes in 0.5 N NaOH, 1.5 M NaCl, and two 30-min washes in the transfer buffer). In both studies the membrane used was Zeta probe (Bio-Rad, Richmond, CA). 

Transfer the proteins to nitrocellulose (Western blot) in transfer buffer (25 mM Tris, 192mM glycine, 0.1% (w/v) SDS, and 20% methanol) for 2h at 0.5A (constant amperes). 

Prepare nylon as in D2, but use transfer buffer instead of 10 x SSC, assemble transfer pyramid and blot as in A6-10. 

After electrophoresis, soak the gel for 20 min in transfer buffer and vacuum-blot to a nylon membrane (4 h) or as above (A). 

Run Criterion gels according to the manufacturer s procedure. Specifically, run them at 200 V until the dye front reaches the bottom of the gel ( 1 h) and transfer them overnight at 4°C (at 30 V) to PVDF membranes using transfer buffer for Criterion blots containing 15% methanol. 

Dunn, S. (1986). Effects of the Modification of Transfer Buffer Composition and the Renaturation of Proteins in Gels on the Recognition of Proteins on Western Blots by Monoclonal Antibodies, Anal. Biochem. 157 ... 

Some changes have been made to the basic procedure over the years. The original procedure as described by Southern (1) used the capillary action of the transfer buffer to provide the motive force to transfer the DNA from the gel to the membrane. Vacuum blotting was first... 

Gels can also be transferred by capillary blotting by a slight modification of the procedure described in Chapter 7. Pretreat the gel with denaturing solution as described above (Section 3.3., step 1) and prewet the membrane (Section 3.3., step 5), then carry out steps 7-12 of the Methods section of Chapter 7, but using 20X SSC as the transfer buffer in place of lOX SSC. Continue from the rinsing step above (Section 3.3., step 16). 

Electroblotting is the most commonly used method of transferring proteins from a gel to a membrane. The principal advantages are the speed and the completeness of transfer compared with diffusion or vacuum blotting. Electroelution can be achieved either by (1) complete immersion of a gel-membrane sandwich in a buffer (wet transfer) or (2) placing the gel-membrane sandwich between absorbent paper soaked in transfer buffer (semi-dry transfer). 

Semi-dry transfer For the semi-dry transfer, the gel-membrane sandwich is placed between carbon plate electrodes. Semi-dry or horizontal blotting uses two plate electrodes (stainless steel or graphite/ carbon) for a uniform electrical field over a short distance, and sandwiches between these of up to six gel/membrane/filter paper assemblies, all well soaked in transfer buffer. The assembly is clamped or otherwise secured on its side and electrophoretic transfer effected in this position, using as transfer buffer only the liquid contained in the gel and filter papers or other pads in the assembly. 

Figure 2 Western blot transfer sandwich assembly for wet transfer (top) and the preparation for transfer using the Mini-Trans-blot apparatus (bottom). The transfer membrane is sandwiched between the gel, filter papers, and support pad, with the transfer membrane facing the anode. The cassette containing the assembled gel-membrane sandwich is inserted into the Trans-blot apparatus containing cold transfer buffer, and the electro-transfer carried out. (Adapted from Bio-Rad Instruction manual.)...

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