Large proteins in Western - (Sep/14/2012 )

I want to analyze some large proteins using WB and I have read different opinions about how to do it.
On one hand I have found that it is improtant to decreasde MeOH concentration to 10% and add SDS in transfer buffer up to 0.1%. On the other hand there are some bibliography that indicate not to change transfer buffer.
Additionally I have also read that I may change voltage and time of transference.

I am confused. Can someone give me some advice?

Thanks you very much in advance

depends on how large the protein is.

you can reduce the acrylamide concentration of the gel

add up to 0.05% sds and 20% methanol to the transfer buffer

transfer for more time (how much more should be determined by trial)

remember that you may never get 100% transfer

here is a useful book:

What's the size of your protein of interest?

I was optimising conditions for the transfer of a ~400kDa protein and had some decent transfer with the following conditions:

5-6% resolving gel (homemade polyacrylamide) - run the 250kDa ladder to halfway down the gel during SDS-PAGE (it's advisable to keep a housekeeping protein on the gel - I used tubulin)

2x Tris-glycine transfer buffer - double the Tris and glycine content. I kept the methanol to 20% (will most likely try to decrease this to 10% as suggested on this forum) and didn't add SDS (might try to add it in the buffer). These 2 additions might make it even favourable.

Wet transferred it overnight 30V followed by another 4 h transfer at 100V. Its important to use a cold room or at least an ice bath because the buffer could heat up (especially the 100V transfer). You can change the ice block to a new one to maintain low temperatures.

Hope this helps!

if you add sds to the transfer buffer then you need more methanol to properly strip the sds from the protein after it has done its job in facilitating transfer. it would be best to allow the transfer buffer to remain at 20% methanol.

Thanks to very much for all your advice. My proteins are not as large (200KDa) so I think that I do not need an over night transfer. I will check it but I will add SDS to the normal transfer.

Thanks again! (hope work ;P)

200 kDa is nothing. Don't worry. You can use normal transfer conditions with increasing a bit the transfer time. For 400 kDa or 600 kDa (tyroglobulin) then we are talking about changing conditions.

Yup agreed with @ascacioc. 200kDa is considered as a fairly transferable size as most ladders have 250kDa as the largest marker. Transfer under normal conditions (10% SDS-PAGE gel transfer for 1h at 100V in the cold room/ice bath...at the end of the transfer, if your 250kDa has fully transferred from gel to membrane, then you know it's definitely transferred) and let us know how it turned out

I wanted to change transfer conditions because after the transference I can see big proteins still in the gel (coomassie staining).
I have tried changing tranfer buffer (10% MeOH and 0,1%SDS) and there are no proteins in the gel (GREAT!!). But now my problem is that the staining of marker proteins have dissapeared so I do not know if the band corresponds to 200KDa (WEIRD!).

I'm suposed to buy another marker with chemiluminiscence.

Thanks you very much for all your help!

criscastells on Thu Sep 20 15:12:28 2012 said:

Yes, the standard protein (all of them) are lost during the transference.