For western blotting, we are using Bio-Rad Trans-Blot® Turbo™ Transfer System by using semi-dry consumables. Although protein marker transfers effectively to the PVDF membrane in 30 mins., more than half of the total protein remains in the SDS-PAGE gel. We tried to optimize methanol concentration in the transfer buffer and changed the glicine with a fresh one but no apparent success we got.
I wonder is there anybody who had similiar experience with this apparatus or do you think our problem is completely different? Any suggestions?
Many thanks in advance.
Hi Erhan,
not all of the protein in the gel SHOULD transfer - a good transfer will transfer ~~20% of the total protein.
even the marker doesn't transfer - what transfers (fast) is the dye.
We did not have much luck with the semi-dry Bio-Rad Trans-Blot® Turbo™ Transfer System when we tried to transfer our protein. My guess is that this system might work for some proteins, but does not work for others. One thing that I noticed is that the membrane and the gel are getting heated much more than in regular liquid western blot system which we usually perform in cold room. I don't know if that was the reason why we could not use this system for our protein, but I thought you might wanted to know that. I would recommend to use the old liquid system for transfer.
Hi there
Although semidry system is fast, requires less reagents but not necessarily as efficient as wet transfer...The difference is more prominent for high mol wt proteins...I guess you also face the problem with high mol wt proteins.....also try to use proper SDS conc in lysis and transfer buffer..denaturation status of protein affects transfer....what memb are you using...Nitrocellulose is better for larger proteins....with semidry transfer lot of things matter including temp during transfer....Anyways the transfer is not always 100 per and if your antibody can detect the proteins on memb that efficiency is not a big issue...
thanks
Hi Erham
We have the same trans blot machine in our lab and always worked well. When we are transferring proteins to PVDF membranes, we use a transfer buffer that contains (among other things) 200ml of absolut methanol and 10ml of SDS (10%) per liter of water.
If you are trying to transfer high molecular weight proteins, try removing completely the methanol from the transfer buffer (we had problems transferring "heavy" proteins and we found that the methanol in the transfer buffer was the problem).
We use a 30 minute program (25V, 1.0 A).
best of luck
Thank you all for your kind interests to my question. To be more clear, I decided to show you a representative image (see below).
We generally work with cell extracts and try to determine expression level of some native proteins which have relatively low level of expression. So, efficient transfer is crutial for me.
Many thanks again.
http://imgur.com/6sRBnBT
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