I would like to detect PKM2 oligomerization state (dimer vs. tetramer) in a variety of cancer cells. Most literature I have noticed use Size Exclusion Chromatography to answer this but Native Gel seems easier/cheaper. The pI of PKM2 is ~ 7.6 so this may not work for this particular protein and may be why no one has had success who has tried it? Has anyone gotten this to work? I am using the antibody from Cell Signaling Techonology (D78A4). I have contacted them and they have mentioned that this antibody should work to detect in Native PAGE as well as it works for IP? Thanks.
You can use variety of biochemical and biophysical methods to address this question:
size-exclusion chromatography (SEC), Blue native PAGE, gradient ultracentrifugation etc. The SEC is the method of choice when working with total or partially fractionated material and detecting your protein of interest via WB. It can be easily calibrated, provides high resolution and allows to run relatively high amount of material, which may be crucial for the detection.
The regular native PAGE, chip and fast technique, may give you some puzzling results, as it will separate based on pI of the protein/pH of the system you use. If you expect your protein being extensively modified (especially, phosphorylated, acetylated or ubiquitylated), then it may appear on the resulting WB (following the native PAGE) as a number of bands, which may not necessarily reflect its oligomerization status.
The Blue Native PAGE technique is better than regular native PAGE and simpler than SEC. But it works well when you do not have to follow with transfer onto membrane, e.g if you work with purified proteins. I used Blue native PAGE with Novex gradient gels in order to separate purified mono-, di- and tetrameric p53 proteins in the presence of Coomassie R-250: worked really good. If you still want to use Blue Native PAGE, you will need to do multiple extensive washes in order to get rid of residual Coomassie R-250 that interfere with transfer.
One more thing: as long as your protein is in crude lysate (or even in partially purified fraction such as nuclear extract or cytoplasm), it might be difficult to show with the high confidence that the change in migration (retention time in SEC) of your protein is not due to interaction with some other co-factors/partners. Use the control along with your experimental sample: mutants that are known to be in only one oligomeric state (dimer and monomer) and unable to form tetramers.
Good luck
You could try two-dimensional PAGE where the first dimension is native and the second dimension is denaturing PAGE. This would likely answer the question of whether there are other binding proteins that result in differences in migration rate. e.g., if your protein is 50 kDa, in the first dimension, you should see two bands - 100 and 200 kDa and in the second dimension the whole thing should run as a 50 kDa species. As Oleg mentioned, R-250 might be a pain to get rid of, from the gel. I used the following protocol to destain my PVDF membrane after transfer.
http://www.sciencedirect.com/science/article/pii/000326979290213Q
Using this destaining method, I could transfer to PVDF and follow with western blotting to detect the protein of interest. If you are still in doubt, you could try the NAMOS assay to be absolutely sure of the oligomeric state of your protein of interest.
http://www.sciencedirect.com/science/article/pii/S0022175907001494
A word of caution though! Results from this assay can be pretty difficult to interpret. I tried this a few times and had to give up after a point because the possibilities seemed infinite. In my case it was a multi-subunit membrane protein. You might be better off if you are working with homo-oligomers.
Good luck
You can just cross link your proteins (using DMP, DSS, BS3 etc) and run SDS PAGE and western blotting. In my experience the simplest and easiest way is just prepare your protein samples in native sample buffer without beta mercapto ethanol or DTT and run them on SDS PAGE and western blotting. You can easily detect which one is dimer or tetramer. Hope this helps you.
Hi Suman, I'm sorry for late response. I do crosslink proteins in cell so that I can avoid losing protein interactions/dimerization during the extraction process. Please find my paper in the attachment for the protocol in method section. After crosslinking, you may need to scrape off the cells into lysis buffer as cross linking agents fix the cells to the plates. Let me know if you have any other questions? Good luck.
Lakshmi
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