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Western blot problems


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#1 postdoc in exile

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Posted 30 August 2010 - 12:30 PM

Hi

I have been trying to do western transfer recently in vain. By far it is not my first, although I am using new equipment that is not familiar to me (BioRad Criterion system). The problem is that I do not see any or very little protein on membrane after transfer (50V for 1 hour in 20% methanol, Tris-Glycine buffer) when stained with Ponceau S. And at the same time something weird is happening to gel, after transfer it is way bigger than before and has also cracked like a desert soil. Buffers that I am using have been used succesfully before, so it is not a question of wrong solutions. Stuff that is happening to the gel seems to point to some sort of discrepancies between buffer system in gel and in transfer solution, although I equlibriate gel in transfer buffer for 15-20 min and prewet membrane and filter paper in transfer buffer. Protein should show up on membrane after transfer, since I am loading 40 ug in every well.. Any ideas/thoughts are welcome.

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#2 laurequillo

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Posted 30 August 2010 - 07:51 PM

Hi

I have been trying to do western transfer recently in vain. By far it is not my first, although I am using new equipment that is not familiar to me (BioRad Criterion system). The problem is that I do not see any or very little protein on membrane after transfer (50V for 1 hour in 20% methanol, Tris-Glycine buffer) when stained with Ponceau S. And at the same time something weird is happening to gel, after transfer it is way bigger than before and has also cracked like a desert soil. Buffers that I am using have been used succesfully before, so it is not a question of wrong solutions. Stuff that is happening to the gel seems to point to some sort of discrepancies between buffer system in gel and in transfer solution, although I equlibriate gel in transfer buffer for 15-20 min and prewet membrane and filter paper in transfer buffer. Protein should show up on membrane after transfer, since I am loading 40 ug in every well.. Any ideas/thoughts are welcome.

Postdoc in exile

Are you using a semy-dry or a wet system for the transfer? With our semi-dry transfer system, we have to do the transfer at 24V tops. Just and idea...
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#3 mdfenko

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Posted 30 August 2010 - 08:54 PM

what is the size of your protein? what is the pore size of your membrane? you may have blown the protein through the membrane.

are you using pvdf? if so, did you activate in pure methanol?

swelling (with cracking) is not unusual but can be controlled, to a degree, with the pressure with which the sandwich is held together.

Edited by mdfenko, 30 August 2010 - 08:55 PM.

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#4 postdoc in exile

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Posted 30 August 2010 - 09:21 PM

Thanks for replies.

My protein is about 55 KDa, but this inability to transfer is insensitive of protein size, it is not the case of smaller proteins being transfered and bigger ones not. I use nitrocellulose from Hybond (the one with reinforced structure, does not crack that easily). And I am using wet system, BioRad Criterion. Gels are BioRad precast ones.

#5 Roo

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Posted 31 August 2010 - 08:21 PM

I know you said the buffers had been successfully used before, but by you or someone else? First, are you sure you are using the right buffer system for running your Bio-Rad Criterion gels. The Criterion gels come in different types (Tris-HCl, Tris-Acetate, Bis-Tris, etc.) To my knowledge, not all of them use the same buffer. You might check the catalog number of the gel against the product literature for that gel to make sure you are using the right buffer to run the gel and then, of course, to transfer it.

We usually use the pre-cast NuPage Gels from Invitrogen. Before NuPage though, I was used to using self poured Tris-Glycine gels. Most of the NuPage gels we buy are Bis-Tris. I didn't check this the first time I grabbed one out of the fridge so I used my "normal" transfer buffer instead of the recommended buffer. Needless to say, things didn't turn out so well. Just double check that you are using the correct buffers for the particular gel type you are using.

If and when you get a gel that isn't completely cracked after transfer, I would suggest staining it with Coomassie instead of using the Ponceau S on the membrane or at least in addition to the Ponceau S. It will tell you whether anything transferred out of the gel or not. This way you know for sure that some protein transferred and it didn't blow through the membrane.

Edited by Roo, 01 September 2010 - 05:59 AM.


#6 mdfenko

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Posted 31 August 2010 - 09:30 PM

you may not have good contact within the transfer sandwich, your sponges may be worn out.
talent does what it can
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