Transfer of large molecular weight proteins - Complete transfer of large molecular weight proteins (Apr/05/2008 )

HI,

I am having problems with wet transfer of 200-250 kDa proteins (membrane proteins). I tried lot of methods but to no avail.
I use PVDF membrane and Towbin's buffer with 10% SDS, 7% gel. I also tried overnight transfer at 30V but I still see some proteins on the gel after transfer. I would really appreciate if I could please get some inputs and suggestions. THanks.

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10% SDS? I think this is not classical Towbin protocol;
better use 48 mM Tris, 30 mM Glycin, 0,037 % (w/v) SDS, 20 % (v/v) Methanol

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Our lab works exclusively on membrane proteins and we use the semi-dry transfer system. IMHO it works much better for membrane protein transfers. If you can borrow one give it a try.

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Sorry for the typing error, what I meant was that I use Towbin's buffer with 10%methanol and not SDS.

I also tried with 0.02%SDS + Towbin's buffer ( 100V for 2hr transfer) and still I see bands on my gel.

Help will be appreciated.

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For wet transfer, we also use the buffer that The Bearer wrote. Are you using a thin or a thick gel? Using a thin gel (I think 0.8mm comb and spacer) might improve the transfer (if I am not wrong). In our hands the wet transfer works better than the semi-dry for big proteins.

Cheers

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Thanks Zek, I'm using 1.5 mm thick gels. I just wanted to know how long you transfer with the buffer which the bearer had suggested? I tried overnight but still I see bands on the gel which obviously didnt transfer onto PVDF . Thanks

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What type of PVDF membrane are you using - single or double membrane? I'd suggest you use the double-layer (which was often used for Edman sequencing), and increase the voltage. PVDF binds proteins very well, so once it leaves the gel, it stays stuck!

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I'm using Immobilon-FL (coz, I'm doing fluorescent detection) from Millipore.

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You can do a quick test. Instead of using one piece of PVDF, use two, and run at the higher voltage. If anything blows through the first membrane, it will be caught by the second one (although I very much doubt that will happen. PVDF binds protein very well, as I said).

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We transfer at 120-150mA for ~20h (for big gels/big proteins) and we use the BioRad immunoblot PVDF membrane.

But I have to say that the very upper bands (near to stacking gel) stay in the gel. So, in your case you get transfer of the small/middle size proteins but not the upper ones? How about your marker? The same problem with that?

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