Membrane blotting

Place several layers of filter paper on a smooth, hard surface, and place a blotting membrane on top (see Fig. 1). Depending on the size of the tissue, use a single- or double-edged razor blade to cut a tissue section 0.2-2.0 mm thick, depending on the particular tissue sample. It is necessary... 

Identify the protein spots as usual, i.e., by staining, autoradiography, gel overlay, or Western blot. In the latter case the gel must be separated from the GelBond foil prior to electrotransfer. For this purpose a film remover (Gorg 2003, Fig. 19) is used The gel is placed on the cylindrical remover with foil down, clamped on an edge, and a thin stainless steel or nylon wire is pulled between foil and gel towards to your body. Cover the gel with the wetted blotting membrane (cf Protocol 2.4.3) and transfer membrane as well as gel to the blotting apparatus. 

Covering the blotting membrane with a suitable capture ligate, e.g., antibody, increases the sensitivity for detecting the corresponding ligand, e.g., antigen. 

Dilute the affinity-purified capture antibody to lO-lOOpg/ml in Soln. A. Incubate a new piece of blotting membrane with the antibody dilution (0.2-0.5 ml/cm ) at RT for 30 min. 

Blotting membranes and TCL plates are processed analogously. Incubate the dry plate or membrane in Soln. B for 5-10 min, dry again, and expose to film. 

B 3. An economical way to block a blotted membrane is to incubate it in a 10% solution of nonfat milk powder. How does this solution function as a blocking reagent ... 

N.B. Always wear gloves when handling blotting membranes to prevent the transfer of protein from fingers onto the membrane surface. 

Staining of blot transfer membranes permits visualization of proteins and allows the extent of transfer to be monitored. In the protocols described in this unit, proteins are stained after electroblotting from one-dimensional or two-dimensional polyacrylamide gels to blot membranes such as polyvinylidene difluoride (PVDF), nitrocellulose, or nylon membranes (unitb3.2). PVDF is the preferred, more universal membrane and is emphasized here however, most stains work similarly on nitrocellulose, and many can be used on alternative blotting membranes. 

Coomassie brilliant blue, 170-171,180,182 plant cell walls, 699-701 (figs.) of proteins on blot membranes, 199-205 silver, 171-172, 180, 182 Standards... 

TOF. see Time-of-flight Trans-Blot membranes, electroblotting and elution, 185-190, 193-197 trans fatty acid determination by FTIR-ATR, 505-511... 

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