I have crosslinked two proteins using typical crosslinking reagents. However, the crosslinked product runs as a smear instead of a tight band. Does anyone have an explanation for this? Does anyone know how I can resolve product into a tight band? Oddly, the protein was SEC purified and eluted as a distinct peak. I usually see smears when a protein degrades. I doubt this as the case in this instance.
Do you know the amount of protein you are running on the gel? It could simply be overloading, in which case running a gel with various dilutions of the crosslinked product could help you find the amount at which you can clearly resolve the band.
What size in the protein in monomeric form? What percentage gel are you running? Maybe a lower percentage gel or a gradient could improve resolution.
Another possibility would be non-specific crosslinking, although the single peak on size exclusion suggests this is not the case.
Eric Simko is right with overloading and also always use fresh APS 10% and not too old TEMED as this makes polymerisation uniform through the gel. This might also give you smeared / not nice bands... You can also cast it the afternoon before the run and let it polymerise in the fridge overnight then you can be absolutely sure it polymerised well.
A good source for failed PAGE gels with explanations can be found here http://www.ruf.rice.edu/~bioslabs/studies/sds-page/sdsgoofs.html just click on the Image and it explains the error..
1. Randomized cross-linking of your protein might end up so many different protein bands with very close proximity leads to smearing on SDS-PAGE gels.
2. Crossl-ink your protein at different concentration of cross-linker to understand the optimum conditions.
3. Reaction pH might control the protein cross-linking as well
4. If you provide me some details of your protein and type of cross-linking your trying i could help you with this. Share the information only suitable for wider public. my details are [email protected].
you can quantify the reduction of the monomer to determine the amount of any specific protein that you are looking for. In case of cross-linking of multi-protein complexes creates difficulties in obtaining clear bands but run as a smear
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