IP heavy chains not in Ponceau, but in final blot exposure (View forum version)



JDSBlueDevl

Posted 21 February 2015 - 12:00 PM

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.


bob1

Posted 22 February 2015 - 11:40 AM

The heavy chain will be easily detected by secondary antibodies, assuming that both your IP and WB antibodies are of the same species. The binding of the secondaries to the heavy chain is very strong and highly specific, hence the strong band.  Not being able to see the HC band by ponceau is reasonably common.