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Can't see my molecular weight markers on PVDF


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#1 Guest_kelly1_*

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Posted 19 March 2015 - 07:24 PM

I have been trying to transfer minigels after running SDS-PAGE this week and I keep having the problem of not seeing my MW bands on the PVDF. I am using two kinds of MW markers, one is from bio-rad, and another is the ultra low molecular weight range marker from sigma aldrich. For both markers, I cannot see the bands on the membrane, and they do not appear on the whatman paper behind my membrane either. I am also hesitant to probe my membrane with primary antibody because it is really hard to obtain, and it would be a waste of solution if there was no target on my membrane I think.

 

just to give you an overview of what I did; I ran samples containing a 1 kDa protein and my markers on the minigel, and then I transferred overnight (16 hours) at 30 volts using transfer buffer containing methanol. I also wet my membrane for about a minute in 100 % methanol before running, and I am pretty sure my electrodes and sandwich were set up correctly. So is there anything else that could be going wrong here ?

 

Lastly, I read another post on here where someone suggested using ponceau red to stain the membrane and check if the proteins were on the membrane. If I only have coomasie brilliant blue available, could I use that and successfully destain my membrane ?



#2 5280

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Posted 19 March 2015 - 08:47 PM

Not sure what you mean with the sample containing a 1 kDa protein. Are these samples total cell lysates? How protein are you loading? Why are you transferring overnight? 

 

I would not stain my PVDF with coomassie. You could stain your gel with Coomassie and see if any protein remains. Ponceau S or Fast Green are pretty common so you should be able to obtain a small amount from another lab to stain your PVDF. . 



#3 mdfenko

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Posted 24 March 2015 - 04:21 AM

what sds-page formulation are you using? for a 1 kDa peptide you should use a 16.5% tricine gel if you are not very concerned with higher molecular weight proteins or you can run a 10-20% gradient gel (tricine).

 

what is the pore size of your membrane? for a 1 kDa peptide you should use 0.2um or smaller and a relatively short transfer time.

 

i used to transfer for ~20 minutes with a semi-dry transfer unit when working with molecular weights in the range in which you are working.

 

what is the methanol concentration in your transfer buffer? it should not exceed 20% (the main purpose of the methanol in the transfer buffer is to strip sds from the protein to prevent it from interfering with binding to the membrane).

 

as 5280 said, coomassie stain can not be easily removed from the transfer and is not recommended for a membrane which will be used for immunoblotting. ponceau s is the dye of choice for this purpose.


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