I'm noticing an interesting curiosity. I'm transferring my 4-20% gel overnight to a PVDF (Immunoblot) membrane in 1X TG buffer only containing 0.1% SDS (no methanol), as this is the only way I've been able to get my high molecular weight protein transferred. Previously, I did the transfer at 30 V, but was noticing that this was leading to a very high current at the end of the transfer. I saw that this transferred my heavy-chain bands very efficiently. Now that I'm using a different power pack, I'm switching to constant current overnight (150 mA). I do not see my heavy-chain bands in the Ponceau stain for this (using 10-15 mL fresh Ponceau solution from Sigma rather than re-used), but when I use a secondary antibody that corresponds with what I used for my pulldown, I get very strong bands where the heavy chains should be (both normal mouse IgG and the IP antibody). It has me a little concerned, but I'm wondering whether I should just regard this as merely an intellectual curiosity rather than a concern. I thought it may be of interest because I had been having problems with pulling down my protein of interest, but that may be due to using a lysis buffer that is not RIPA (when I tried it a while back with RIPA, but with the old transfer method, I was able to see pulldown). However, now I'm getting the same thing when I switched back to RIPA.
Edited by Hic5Prostate, 21 February 2015 - 12:01 PM.