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dot blot works, western doesn't - HELP!


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#1 Adeno-associated virus

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Posted 21 September 2010 - 01:01 AM

Hi,

my problem is described in the title. It is a membrane protein. The antibodies are fresh. I use NO reducing agents (like B-mercaptoethanol or DTT) in Western blots. I equilibrate the gel in semi-dry transfer buffer for 30 minutes. I transfer in 20% methanol 0% or 0.02% SDS semi-dry transfer buffer. I incubate with antibodies and wash for the same amounts of time as for dot blot. Nothing seems to be working. I'm pretty sure it's not SDS destroying my epitope, because I also did a dot blot where I pre-incubated my protein with 1 x running buffer before blotting on the membrane and I got the signal.
Any suggestions anyone?

Thanks,
Andrzej

#2 Prep!

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Posted 21 September 2010 - 02:38 AM

hi tere Andrzej .. it wud be most helpful if u can give in more details like the run conditions and transfer and development conditions.. are u sure that the transfer is efficient?? have u checked with staining the transferred gels... or have u done a ponceu on the blot before developing with antibodies??!!! wat do u mean by same time washes... cause if u have washed more then the antibodies might also have been washed off...
a little more details will be definitely helpful!!!

Best luck!!

Edited by Prep!, 21 September 2010 - 02:38 AM.

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#3 Adeno-associated virus

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Posted 22 September 2010 - 10:36 PM

hi tere Andrzej .. it wud be most helpful if u can give in more details like the run conditions and transfer and development conditions.. are u sure that the transfer is efficient?? have u checked with staining the transferred gels... or have u done a ponceu on the blot before developing with antibodies??!!! wat do u mean by same time washes... cause if u have washed more then the antibodies might also have been washed off...
a little more details will be definitely helpful!!!

Best luck!!


Hi there Prep!

My running conditions are 90 minutes @ 120V. The marker separates nicely. The running buffer contains 0.3% Tris, 1.44% glycine, 0.1% SDS. Transfer for 45 minutes @ 25V. Transfer buffer: 0.582% Tris; 0.293% Glycine; 0.0375% SDS; 20% methanol. Ponceau on membranes also shows very nice and even lanes of proteins. By same times of washes, I mean: block overnight in 3% BSA in TBST (1.2% Tris, 0.9% NaCl; 0.1% Tween-20; pH = 7.5); incubate with primary antibody prepared in blocking solution for 1 hour @ RT; 3 x 5 minute wash with TBST; secondary antibody in blocking solution 1h @ RT; 3 x 5 minutes wash with TBST. All of these incubations/washes are identical in dot blot and Western blot, and dot blot works, but Western doesn't. I did stain the gel after the transfer and only some huge proteins >250kDa were not transferred efficiently, but I'm not interested in those.

Is it possible that my epitope is sensitive to SDS or methanol and therefore gets destroyed while being run in the gel or transferred to the membrane? (I use no reducing agents in any of the protocols).

Thanks!
Andrzej

#4 Prep!

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Posted 22 September 2010 - 11:48 PM

Going by what u are describing here... the best thing would be to check if SDS is affecting your protein epitope!!! I say this even though u have made the dot blotted sample in SDS running buffer... but the protein is going to be in the presence of SDS for a long long time!!! Anyways to check if SDS is affecting or not u can do a native PAGE and for methanol u can simply eliminate the methanol and do the blot and see...

But i wud suggest first to go thru the literature of the antibodies or contact the vendor and describe the problem first before doing any more experiments!!

Best luck!!
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#5 Adeno-associated virus

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Posted 23 September 2010 - 04:27 AM

Going by what u are describing here... the best thing would be to check if SDS is affecting your protein epitope!!! I say this even though u have made the dot blotted sample in SDS running buffer... but the protein is going to be in the presence of SDS for a long long time!!! Anyways to check if SDS is affecting or not u can do a native PAGE and for methanol u can simply eliminate the methanol and do the blot and see...

But i wud suggest first to go thru the literature of the antibodies or contact the vendor and describe the problem first before doing any more experiments!!

Best luck!!


Thanks for your reply Prep!.
I was thinking the same thing - do a Western with no SDS or ethanol whatsoever. I did a dot blot, where I pre-incubated the protein with 0.1% SDS and also with running buffer for 2 hours before blotting on the membrane. The result was strange, because SDS alone decreased the dot blot signal, but running buffer, which also contains 0.1% SDS - did not. Another buffer that decreased the signal during this incubation was transfer buffer.
I can't really contact the vendor, because the atibodies I'm testing are actually polyclonal anti sera raised in house :)
Thanks for your suggestions again!
Andrzej

#6 mdfenko

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Posted 24 September 2010 - 07:51 AM

what is the size of your protein of interest? maybe it is blowing through the membrane during transfer or not moving at all. have you stained the gel after transfer?
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