It depends on each particular protein and the conditions of WB used. Each protein might behave differently depending on the molecular weight, charge, hydrophobicity, conformation, the presence of disulphide bonds, duration of transfer, and so on. Probably, 100% transfer is hard to achieve for most of proteins. Under optimal conditions (and depending on the MW of the protein and other parameters) you can expect the range from ~ 80% to 95% transfer efficiency. The higher MW of your protein, the lower the transfer efficiency. You can always stain your gel after transfer with Silver staining to see how efficient your transfer was.
It depends on each particular protein and the conditions of WB used. Each protein might behave differently depending on the molecular weight, charge, hydrophobicity, conformation, the presence of disulphide bonds, duration of transfer, and so on. Probably, 100% transfer is hard to achieve for most of proteins. Under optimal conditions (and depending on the MW of the protein and other parameters) you can expect the range from ~ 80% to 95% transfer efficiency. The higher MW of your protein, the lower the transfer efficiency. You can always stain your gel after transfer with Silver staining to see how efficient your transfer was.
In the pre-ECL days, IBs were visualized colorimetrically using alkaline phosphatase-conjugated secondary antibodies and BCIP/NBT. This technique could reveal immune complexes on both sides of the membrane. In fact, it is possible to over-transfer a protein - pushing it right through the membrane. This happens more rapidly with lower molecular weight proteins and very acidic proteins (assuming Tris-Glycine transfer buffer).
Hi Sakthi Vel
I agree with Dr. Victor. 100% of efficacy in protein transfer is very hard to achieve and depends of the intrinsic structure of the protein and the transfer conditions. But in the most of case it is not necessary. There are different detection systems able to detect very low concentration of molecules target so, incomplete transfer maybe no be a problem.
Thank you very much for your answers, Viktor Y Butnev and Oscar Otero. Thank you Professor (Bruce Allen) for your reply.
There are many factors that influence the efficiency of transfer in western blotting results these include the membrane used ,the type of transfer method used the size and nature of proteins being used small proteins less than 60 KDa are usually more efficiently transferred than larger ones also the detection and probing wet ,semi- dry and dry electroblotting manual or automated etc.These factors make it difficult to obtain 100% transfer ultimately if you are lucky with the size and nature of your protein and you set the right conditions you can get 95% transfer. Good luck
Thank you very much dear Professor (Abdelrahim Ahmad Hunaiti) for your kind and beautiful reply. Now I really got some quantum of knowledge from your answer ! and thank your for wishes. I hope your are doing well and good, I wish you the same.
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