Western trasfer and blotting troubleshooting

[shifali chatrath]

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%  
>> methanol.
>> 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... :))

>Science" ;-)
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Shifali Chatrath
Graduate Student
Protein science Lab
Dept. of Biological sciences
National University of Singapore
Singapore
+65-65161210
+65-96393449