Western trasfer and blotting troubleshooting
(by shifalich from rediffmail.com)
Tue May 5 03:28:59 EST 2009
On Tue, 05 May 2009 00:54:40 +0530 wrote
>Am 03.05.2009, 23:47 Uhr, schrieb shifali chatrath
>> I transfered the protein on 12% Tris -Tricine gels to Methanol wetted
>> PVDF membrane using semi-dry apparatus at 300A, 9V for 40 min.
>> Transfer buffer: 48mM Tris,Glycine 39mM pH(8.9) without adjusting,+ 20%
>> After transfer, markers on one side(first lane) got beautifully
>> tranfered but the other side of the gel (last lane),got diffused, can
>> anybody suggest why?Anyhow, I proceeded for blotting.
>There is little point in proceeding with ab staining, if the transfer did
>not work. After wetting the PVDF with MeOH you need to transfer to
>blotting buffer, as MeOH is not conductive. The blotting sandwich needts
>to be assembled without air bubbles. Are the electrodes of the semi-dry
>blotter ok (not unevenly worn)?
How to check if the electrodes are OK?
I did soak the membrane in transfer buffer before transfer.
>> 3X washes in PBS. Then I added 2 Ab,anti-mouse preadsorbed human, 1:5000
>> in Blocking buffer, it got pptd. on ice and i notice only after adding
>> on blot.
>Well, it shouldn't, so you must have done something wrong. The precipitate
>is probably not the antibody, as the concentration is too low to cause a
>visible precipitate. Redo the blocking buffer, making sure that you get
>the right chemicals in the correct amounts. If that doesn't get rid of the
>precipitation, leave the compounds out one by one to find the culprit. It
>could be some contaminant, or, in very rare cases, a bad batch of
>chemicals (Sigma once send me "SDS" that was water insoluble).
because of milk products in blocking buffer which can be dissolved at 37,I mentioned this because after secondary Ab incubation, 2 of the pretsained markers were missing from the membrane.So, I was wonderin if temp. is the reason for loss of proteins frm. the membrane or wht?
I understand that the pptn. is not because of Ab, but
>Alternative option: consider publishing in the "Journal of Irreproducible
Is there any such journal? I have many manuscripts to publish then... :))
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