I am doing western blotting for sample after PFA cross-linking. And, indeed, I see something which looks like cross-linked band after PFA, but it MW is ~30 kDa smaller than expected.
I would like to understand - is it common and expectable?
I would assume that electrophoretic mobility should be affected by cross-linking, because cross-linking will prevent SDS-denaturation. But it would be nice to have some examples of similar cases.
Thanks!
Hi Arseniy,
After cross linking, are you running the sample in a gel system which is denaturing (SDS0PAGE) or native, (Blue Native Electrophoresis)?
After cross linking, in the BNE electrophoresis, you should see a shift to higher molecular weight of the protein of interest. If you are using denaturing conditions, not sure how the complex is preserved?
Best,
Hediye.
Hi Hediye,
Thank you for you answer!
The question which is the most unclear for me, is how big this shift should be? I expect 75 kDa shift, but observe only ~45 kDa shift.
I am trying to do it in denaturing conditions (with standard SDS-PAGE).
As I understood, the formaldehyde will made cross-links between different protein chains in the complex. As soon as formaldehyde will made covalent bonds, the chains still will be connected after denaturation by SDS.
I am basically trying to reproduce the method from this paper, with some modifications:
Article Optimization of Formaldehyde Cross-Linking for Protein Inter...
Post cross-linking, the sum of the proteins linked by cross-linker is what you should expect to see. In practice however, this is more complicated since cross linking is not a clean experiment and since there are intra- (within the protein) as well as many mono-linkages formed as reaction products.
So, if you are observing a cross linked product smaller than expected, is that because you already know what it is linking with?
Hediye.
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