I am working with several protein-proten and protein-heparin complexes. After the complex formation in the tube I can see a shift in gel filtration column but the complexes are not stable enough for analysis in SDS-PAGE im non reducing conditions. Can you please suggest me a good cross-linking protocol in order to fix my protein-protein protein-heparin complexes allowing me to analyze them on SDS-PAGE?
Many thanks
Gluteraldehyde is a common protein crosslinking molecule.
http://www.biotechniques.com/multimedia/archive/00037/BTN_A_04375RV01_O_37190a.pdf
There is also a handy cross-linking handbook available from Thermo as a PDF: https://tools.lifetechnologies.com/content/sfs/brochures/1602163-Crosslinking-Reagents-Handbook.pdf
Hi Giovanni, if your proteins are in close proximity, you could try the zero length crosslinker EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride
https://www.lifetechnologies.com/order/catalog/product/22980
I had very satisfactory results with the amine crosslinker BS3. I liked the fact that it is water-soluble.
I agree with Glutaraldehyde cross-linking being effective.
DOI: 10.1016/j.ab.2007.10.027
EDC is good for crosslinking peptides. If you go this route I would recommend using EDC sulfo-NHS. sulfo-NHS is more water soluble. I have used the attached protocol to make amide bonds. Gluteraldehyde is less specific which is why I would recommend this if you don't care which peptides/functional groups are being crosslinkined. If you used EDC/sNHS, make sure you verify pH is correct. The reaction is very pH sensitive (from experience).
http://www.researchgate.net/publictopics.PublicPostFileLoader.html?id=5328fed0cf57d7160f8b4592&key=e0b495328fed0c892d