Transfer Issues to PVDF

[Kaitou Kid]

Originally Posted by danfive

Try borrowing a power source or asking anyone about limiting the Amps.

If you are staining only one hour I suggest Imperial Protein Stain from Pierce, not the classic coomassie blue (that's usually an overnight stain with an overnight destain).

I hope you already know the amount of protein you are loading to each well, and perhaps a logical explanation to your different staining patterns is that the transfer gel had its excess SDS washed off (through the procedure) and thus stained more efficiently (that is if you also stained 1 hour etc).

hi danfive

You are absolutely right. The SDS was washed off by the methanol during the transfer, so I guess the gel wasn't washed enough. I am pretty sure SOME protein transferred over, but I just ordered some Ponceau S solution to stain the membrane and see if transfer occurred.

I'll let you guys know how it goes. As far as the actual western goes, using these films produce a glowing membrane and dark patch on the film, so I'm going to have to dilute the secondary antibody by 100-1000 I guess.

[Kaitou Kid]

Good news guys.

I bought some ponceau s and stained my membrane after transfer at 15 V for 45 minutes.

BANDS WERE THERE YES!!!!!

Thanks for all your help guys.

Next step is to get this WB working .... haha,. Best of luck to all your experiments.

FOR ANYONE WHO HAS THE SAME PROBLEM:

- as people have told me in this thread, because protein is still in the gel doesn't mean that NO protein transferred.

- check membrane with some reversible stain if you're not sure

- once you're sure, just keep the same run time/volt etc.

[biohou]

Hi guys,
our lab used to transfer at 0.8mA/cm^2 for 2 hours, Most importantly we keep 4 Volts at first .for example ,i have a 4*7.5 cm^2 gel ,i will transfer at 24 mA for hours and keep 4 volts.