transfer problem - i cannot transfer proteins to a pvdf membrane (Aug/19/2008 )
Hi.
I've just started to use semi-dry western transfer system.
The problem is all the proteins in my gel are not transferred.
I found that the current is very low (10 mA) though I set 100 mA for constant current with 24V for maximal voltage.
What would be the problem?
I'm using a PVDF membrane, and the transfer buffer contains Tris Gly and 5% methanol.
Although I already read the very similar topic in important topics, but my problem looks a little bit different.
Thank you in advance for your kind replies.
Are the gel, the membrane and the papers exactly the same size?
Oh.. I didn't know this could be a reason.
I tried to transfer two mini gels into one membrane, and there is a gap between gels.
Thanks a lot
I've just started to use semi-dry western transfer system.
The problem is all the proteins in my gel are not transferred.
I found that the current is very low (10 mA) though I set 100 mA for constant current with 24V for maximal voltage.
What would be the problem?
I'm using a PVDF membrane, and the transfer buffer contains Tris Gly and 5% methanol.
Although I already read the very similar topic in important topics, but my problem looks a little bit different.
Thank you in advance for your kind replies.
use 20% methanol and some traces (0.037%) of SDS
Agree with the Bearer.
Do you presoak your membrane in methanol, and then in the transfer buffer?
How many wattman do you put at each end of the sandwich?
Do you presoak your membrane in methanol, and then in the transfer buffer?
How many wattman do you put at each end of the sandwich?
Thank you very much, both of you.
My transfer buffer contains 5% methanol and 0.1% sds.
And for presoaking I just use the transfer buffer. Isn't it enough?
My labmate said higher methaol in buffer may prevent the transfer of higher molecular weight proteins. Mine is around 50kDa.
If you tell me the function of methanol in transfer, I'll really thank you.
It is essential that you prewet the PVDF membran in 100% Methanol for just some 30 secs or 1 min, otherwise it will not be hydrophil and let the proteins stick to it. Just transfer buffer is not enough! After prewetting you put in in transfer buffer to adjust the buffer system, let it stay in there for at least 5 min. You can go up to 20% Methanol in your transfer buffer.
And be careful to have (as Minnie mentioned before) no "bridges" between the whatman papers, which don't touch your gel. The current could go around your gel and you will not get good transfer. Thats why everything should have the same size, whatmans, gel, membrane. And everything must be wetted in the same buffer, gel also.
also, don't exceed 0.05%sds in the transfer buffer. it is needed to transfer large proteins but can interfere with the protein's binding to the membrane.
that's where the methanol comes in. one of the functions of the methanol is to strip sds from the proteins to allow them to bind to the membrane.