Western blotting of transforming growth factor beta 2. Optimization of the electrophoretic transfer.
The highest reported detection sensitivity of reduced monomeric transforming growth factor (TGF) beta 2 by Western blotting is 50 ng. The biologically active TGF-beta 2, which is the non-reduced dimeric protein, is even more difficult to detect by this technique. The low sensitivity is due to the poor electrophoretic transfer of the protein from the polyacrylamide gel to the blotting matrices under standard transfer conditions. By studying the effects of different blotting matrices, transfer cells, blotting buffers and their methanol content, current settings and the electrotransfer time, we have established optimal conditions for the blotting of this protein. The pH of the buffer and the blotting time are the most important factors which influence the electrotransfer yield. Optimal transfer of the TGF-beta 2 protein was achieved on PVDF membrane with semi-dry transfer for 4 h at 9 V, using 10 mM CAPS, pH 11.0, containing 5% methanol as transfer buffer. Combined with the use of a commercial antibody and an immunoblot assay kit, our optimised blotting method can detect 2 ng of the dimeric TGF-beta 2.
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Biotechnology Department, Ciba-Geigy Ltd, Basel, Switzerland.
Cerletti
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D015153:Blotting, Western / Q000379:methods*; D002021:Buffers; D008567:Membranes, Artificial; D000432:Methanol; D011145:Polyvinyls; D011994:Recombinant Proteins / Q000032:analysis; D012680:Sensitivity and Specificity; D016212:Transforming Growth Factor beta / Q000032:analysis*; D005465:Fluorocarbon Polymers
D002021:Buffers; D008567:Membranes, Artificial; D000432:Methanol; D011145:Polyvinyls; D011994:Recombinant Proteins; D016212:Transforming Growth Factor beta; C024865:polyvinylidene fluoride; D005465:Fluorocarbon Polymers