I'm using SDS-PAGE to try to detect histone 3 lysine 27 trimethylation (H3K27me3) using a ChIP grade monoclonal antibody from Diagenode. I'm loading 15ug of protein from U937 nuclear extracts, a 15% gel, 0.2um PVDF activated in methanol, and have tried various transfer times. All blots come out looking 'blotchy' (see attached pictures), which has never happened before. Does anyone have any input as to why this could be happening, and what I could do to get a good blot?
Many Thanks.
I've never seen this sort of artifact before. What are your transfer conditions? As histones are basic, have you tried a CAPS buffer at pH 11 (10 mM CAPS-NaOH, pH11/10% MeOH)?
I haven't either, which is why I'm finding it so perplexing. We have transferred for 2 hours at 100v which I normally use for larger proteins with no problems, but because the histone mark is so small, have also tried 30 minutes. We use BioRad's TGS buffer with either 20 or 10% methanol. PVDF membrane, which again, we have not had issues with before and is supposed to be better for smaller proteins.
Bio Rad recommend CAPS buffer for proteins >150kDa, but have you had success using it for smaller proteins too?
Many thanks for your input thus far.
We've been using the CAPS buffer for a 49-kDa, mildly basic protein with good success. I will have to verify with the lab as to the voltage/time. But, I think its 100V for 90 min at 4 deg C in a BioRad Mini Protien cell. For this protein, we are using nitrocellulose.
I admit I've never seen anything like that, it looks like an inkblot test. Maybe it's the gel? I run 4-20% gels all the time for histones and they always turn out beautifully as long as I sonicate really well. Do you soak the gel in transfer buffer for a few minutes to let it equilibrate? You might want to do a Ponceau S on the blot right after transfer to see if it's a problem with the transfer. If that looks all messy, move back and see if a Coomassie stain on the gel gives you a normal array of bands.
- Badges
- Science method