Hi everyone,
I am a beginner in western blot so I really need your help. I often transfer protein to PVDF membrane with these conditions (without caring about protein size) : 80V, 0.2 A, 300 W, 3 hours. But I consider if proteins can go throughout PVDF or not ? For instance, how long do you do transfer step with protein 110kDa or 43kDa ?
Thank you so much,
Lebanam
I don't know they type of blotting system you use but your settings seem to be reasonable for transfering proteins from 20 kDa - 120 kDa. When you want to transfer smaller proteins you should reduce the transfer time by approx. 50-60 % as they can indeed pass the transfer membrane and become lost. For bigger proteins you should decrease the percentage of your gel to increase the protein mobility.
Don't hesitate to ask if you want to know more about Western-Blotting.
Cheers, Christian
For proteins of the size you mention, i.e. not too small, I think that passing through the membrane shouldn't bee too big an issue. However, you may want to look into what membrane you are currently using, as the PVDF western blot membranes come with different pore sizes (typically 0.45 or 0.2 um). Usually 0.45 is fine, but if you suspect you are having problems with proteins leaking through you could try using the smaller pore size membranes instead.
You can also do a simple test of protein blow-through by placing a second membrane behind your primary membrane. Process the second membrane the same way as the first and you'll be able to see/capture any "lost" proteins.
80V, 0.2 A, 300 W, 3 hours
"0.2 A (hold), 2 hr" this condition is enough for transfer.
If your result is not certain, you willl need to check all things for transfer, e.g. buffer, PVDF membrane, cassettes, etc.
Good luck!!
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