We use this antibody on unheated samples: Recombinant Anti-VDAC1/Porin + VDAC2 antibody [EPR10852(B)] - Mitochondrial Loading Control (ab154856). It is anti-rabbit, has a molecular weight of 37 kD.

Hi, I use beta actin for the unheated sample as as well. But in this way, you may need to increase the concentration of primary antibody. (In my experience for beta actin antibody 1:10000 dilution for heated sample ; 1:1000 for unheated sample works well)

Try to apply total protein normalization instead of HKPs...This approach may also validate the experiment...

I recommend total protein normalisation like İsmail Emir Akyildiz . If however you need to use a housekeeping protein, using un-heated samples will not (or is at least highly unlikely to) affect your results. The SDS in the sample buffer is what does the actual denaturing, heating just speeds this up and is unneccessary in most cases. For some proteins, especially membrane proteins, using high heat can actually promote aggregation, so leaving at room temperature or 37 degrees C for a while to denature should work fine. There is an increased risk of proteolysis (as any proteases are not going to be destroyed by high heat), but protease inhibitors can be included in the sample buffer to reduce this. Let us know how you get on.

Samuel Davis

Is it your personal experience regarding high heat promoted aggregation or literature data? Could you provide pdf file or link? I have some problems with detection of cytochrome c, it seems that most of protein polymerized or aggregate

I use beta actin as house keeping gene when using samples with and without heating for westerns.