I am currently trying to detect the collagen 7 (C7) trimer protein (~900 kDa) in its native form via western blot - it is made up of three C7 monomers of roughly 300 kDa. I have already detected the monomer using denaturing western blot conditions, however I am now trying to establish a western blot protocol to detect the C7 trimer, and therefore I need to use native conditions. So far I have had two attempts without any luck. An overview of what I have tried so far is as follows:
- Sample buffer: Native Tris-Glycine sample buffer.
- Gel electrophoresis conditions: Tris-Glycine Native Running buffer using 3-8% Tris-Acetate gels, 150V for at least 3 hours
- Transfer conditions: Transfer buffer contains 10% methanol, 0.1% SDS. I have tried 1hr and 1.5 hr transfer at 100V to nitrocellulose membranes (0.2um pore size)
A couple of questions I have:
1. Should I be heating my protein samples at 95degC for 5 minutes before gel electrophoresis, or is this only necessary under denaturing conditions?
2. Should I include SDS in the transfer buffer?
Any recommendations or suggestions about how I can get this western blot to work will be much appreciated!
With such a large complex, it is likely that the pore size of even low-percentage polyacrylamide is too small to let it enter the gel. You may perhaps consider agarose gels. As to you questions, 1) there should be no need to heat the samples. this may only lead to denaturation and precipitation; 2) incluson of SDS in the transfer buffer shouldn't harm.
What is the PI of your protein? You would need to adjust the pH of your running buffers.
As suggested you may need to increase the pH of your running buffer and gel buffers depending on pI, as the sample may not enter the gel. Tris-glycine I'm guessing is at pH 8.8. As Pierre has suggested a 900 kDa protein may not enter the gel even at 3% acrylamide. You could try using less crosslinker (Bis-acrylamide %) in your gels as Im presuming your ratio is 37:1? This would increase the pore size and may help with separation. You can buy in acrylamides at different ratios or ready made gels.
I would perhaps try the transfer buffer without methanol as this can shrink the pores and for large proteins this may be a problem in transfer or increase transfer times.
Your native loading buffer also needs to be at the same pH as the running buffer. For Native PAGE you should not be heating samples.
I don't think PAGE is suitable to analyze a 1 Mda complex. Proteins migrate much slower than by SDS-PAGE and your complex quite large, probably too large for PAGE analysis to be helpful . Not sure what hypothesis you're trying to test , but I would consider other technical approaches to tackle it.
My experience with PAGE was with BSA (67 kDa), which forms weak oligomers and the run took overnight using conditions similar to yours (slightly more basic pH) just to see the monomer halfway through a 4% gel. Quite skeptical your experiment will be informative given the pI/pH similarity and the mass of your protein. Again, I would look for alternative approaches if I were you.
Hello Alex,
We run routinely high resolution clear native electrophoresis to analyze mitochondrial supercomplexes. We pour the gradient gels (imidazole, aminocaproic acid based). Proteins with a molecular weight of about 1000 kD will enter the gel. Do not boil/heat your sample, as they will be denatured.
For protocol see the attached paper.
As for transfer of a native gel: We have SDS and methanol in the transfer buffer. Our transfer conditions are wet transfer at 25 V overnight in the cold room. Our experience with high voltage wet transfer (90 V for 1.5 h) is, that it works not well at all.
Also consider, not every antibody that works well in a denaturing western blot will work after transfer of natively separated proteins.
Good luck!
Good points Gisela. I stand corrected! Forgot about the mitochondria blue (and clear) gels. Definitely worth a try for collagen complexes.
Thank you all for the informative responses! The consensus seems to be changing the pH of the buffers Sébastien Soubeyrand Junjie Shao Ian R Wilkinson
A slight correction for my previous answer - the PI of collagen 7 is 5.9, not 7. Given this, would you still recommend changing the pH of any of the buffers?
Thanks in advance!
It's not just a matter of pH. The buffer system is also important. Both Blue and Clear native gel systems use completely different buffer systems. I would stick to those buffer systems, optimized for mitochondria complexes analysis but that could be optimized for your application. The Blue native gel system has the advantage of negatively "charging" your protein, which will speed up your migration and increase your resolution (most likely).
https://pubmed.ncbi.nlm.nih.gov/17406264/
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