Hi guys, so after blotting the membrane, and checking for total protein content, I washed the membrane for 20 mins in blocking buffer to remove ponceau s stain, then washed in distilled water for 5 mins, left to dry overnight, and froze it the following day at -20 degrees. The secondary antibody I needed for my work was just delivered today, so now I am wondering what is the best way to proceed with the membrane before blocking it, applying primary antibody, secondary antibody, and reagent. Many thanks
I must say we never freeze our PVDF membranes. We simply dry them after Coomassie and destain/reactivate them in methanol. I would suggest it should work for you too. Simply a minute in methanol, until membrane is not hydrophobic, then quick wash with TBST and proceed with blocking as usual
yes...i dont think freezing was required. However, you may get back to room temperature and activate it in methanol for a minute and then proceed to blocking, primary and further.
Hi, I freezed my membrane too at -20°C, after primary and secondary antibodies.. Now I need to do the stripping in order to repeat a primary antibody. Should I reactivate the membrane in methanol before stripping?
Thanks a lot for answering
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