3 replies to this topic

[gyma]

I have never had such a problem before until recently I found I couldnt get efficient transfer after SDS-PAGE. This is what I used to do:
1, soak PVDF in methanol for less than 1 minute and pour transfer buffer in it and shake until everything is ready, usually several minutes.
2, remove the gel and put it also in transfer buffer.
3, prepare semi-dry transfer apparatus, pour some transfer buffer, put 3 filter paper, add more buffer.
4, put PVDF on the paper, then gel, then another 3 paper, transfer buffer and roller to remove bubbles.
5, 10V, 1.5~2h.

I know it might be different from your protocol but this protocol is always ok for me, even for a protein >200kD. Now the problem is, after transfer, most marker bands were still there (I did ECL anyway but the result was no good). It is strange that marker bands with bigger size were almost successfully transferred but smaller ones were not. I also tried different transfer apparatus and no improvement.

another thing needs to be mentioned is that I just made new 10X transfer buffer stock and this problem seemed to occur after that. Here is the recipe which I always use. 10X: Tris base 37.85g, Glycine 180.175g, add water to 1 L. For 1X, I use 50 ml 10X buffer, 100 ml Methanol and 350 ml water.

It seems to be the problem of my transfer buffer but I dont know whether my recipe is ok or I made some mistakes during preparing the buffer. please tell me if you know what's wrong. thank you.

[almost a doctor]

The concentration of Tris and Glycine seem a bit too high compare to the most standard buffer,  also, did you check the pH of your buffer?  

These are the 2 things I can think of, particularly the pH.

Also, do you check the transfer by staining the membrane?

Edited by almost a doctor, 15 September 2011 - 07:29 AM.

[gyma]

almost a doctor, on 15 September 2011 - 07:29 AM, said:

The concentration of Tris and Glycine seem a bit too high compare to the most standard buffer,  also, did you check the pH of your buffer?  

These are the 2 things I can think of, particularly the pH.

Also, do you check the transfer by staining the membrane?

thanks for your reply.
I got a little improvement for this problem. I made new buffers of both composition and also used buffers from my labmates to check the transfer. same transfer apparatus but different power supply, 10V, 1.5h. It turned out that all the buffers were fine. I couldnt see remaining bind by naked eye but after CBB staining there are still faint marker bands in the gel, mostly small-size bands. But it was much better. So I think maybe there was problem with the power supply.
however, there is still one thing I am not sure. the remaining marker bands look like a little bit more than before. Is that normal or there should be nothing. I used kaleidoscope marker from Biorad.

I didnt check the pH because it is not mentioned in the recipe. what should it be?

[gyma]

in the case of semidry, buffer without methanol works better.
for large protein transfer, I used buffer containing 0.05% SDS and 5% methanol and it worked well for protein >200kDa. However, it was not reproducible and foam problem is also annoying.