2 questions in 1....about transferring large proteins...and blotting - (Dec/10/2006 )

Hi Everyone....I read through the postings and I don't think my questions have already been posted...so here goes....

I am using a semi-dry transfer system and I am transferring a 121kDa protein....I have a tried a variety of milliamps and times and none seem to be successful. I am using PVDF and an a buffer with 10% SDS if either of those make a difference??? So I am wondering if there are any suggestions out there???

Secondly....when blotting with 2 antibodies on the same membrane...can you do them simultaneoulsy?? I couldn't see why not...but maybe some interference between the antibodies...so I figured maybe consecutively is better?? One is a 121 kDA and the other is only 52kDa.....

Hopefully some tips out there
TX
Ames

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The amount of SDS is too high in my opinion: we use 0,02% SDS in our lab. Try using also 10% MetOH.
For the immunodetection: if the Abs don't crossreact with each other, you can use them together. Personally, I prefer to cut the membrane in a lower and upper part and probe the two parts with two different Abs.

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I agree with dnafactory

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Thank you very much...I will give that a try.
Cheers

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Someone just mentioned that wet transfer is better than dry...is that true??
Ames

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It depends on the apparatus and the MW of the proteins. For small proteins, you could have some problems if you use too high voltage, because the current will be too high and the small proteins will pass through the membrane. I found the dry blot better for my purposes in the past but I'm using wet blot now

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I can tell you my little experience: I work with a 50 KDa protein and first I did wet transfer at 400 mA for 1 hour and 30 min and always worked well although I think that perhaps with less time it would have also worked fine. Some time later, I tried semidry (BioRad unit) and now I do it at 15 V for 30 min and it has always gone very well (so I´m still wondering... why I didn´t try semidry before? ) So, I will do semidry because I save a lot of precious time and buffer Thus, from my experience I think the best is to try in each case and decide then

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Thank you...I got the results today
And the MWmarker transferred mostly...but then when I stained the gel with Coomassie there was still lots? of protein there....not sure if it is supposed to all be gone?? Then I had continued with the membrane and it was no good....I should have stained the membrane after but didn't.... So I am going to try the wet tomorrow....and hope it goes well.......I will let you know
Ames

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