Hello,
I use homobifunctional crosslinkers to form protein gel. I am currently using 4-nitrophenyl carbonate leaving groups for protein crosslinking. I dissolve cross-linker in DMSO and slow add the crosslinker in protein solution (pbs (pH 7.4) buffer). I expected to get multiple clear bands in sds-page. To explain my question better, I attached my sds-page picture. Right next the ladder is RNAse A without crosslinkers, from the third to the last, I increased the amount of cross-linkers.
The third lane and the last lane, which are supposed to be containing the least, and the most amount of crosslinkers showed band smearing. but, from the fourth lane to the seventh lane, it seems like proteins are not cross-linked at all.
My first question is, is the rate of adding crosslinkers can affect the crosslinking efficiency?
Second, is it possible that quick addition of cross-linkers prevent proteins from intermolecularly crosslinking each other?
Second, why do I get band smearing in my band? Could I say proteins are crosslinked, even if I get band smearing in my sds-page?
Getting a ladder type pattern after SDS-PAGE is indeed an indication of X-linking. Do not assume that you get a single species of cross-linked products. Because your X-linker will generate multiple intra and intermolecular bonds, your results are typical as proteins can't adopt fully denatured rod-like SDS/protein complexes. Using less concentrated crosslinkers may help to reduce diversity in your sample. Time is obviously an important parameter.
Good luck!
Smearing on gel can be caused by the presence of salts ( eg. from buffers) in the sample.
It is normal to obtain a smear on gel when performing non specific protein crosslinking. As stated before the crosslinking process generate multiple products since you have many different degrees of oligomerisation. To get specific crosslinking you need to use a strategy applied in solid phase peptide synthesis. I mean having the two functions you wish to crosslink equipped with the same protective group whilst other possible reacting functions are blocked with a different protective group, it is very common in directed disulfide bond formation for example. But if your objective is to have a randomly crosslinked gel then having a smear on your SDS-PAGE means you have successful polymerization. Finally, if smearing is due to salts you can simply pass your sample on a desalting PD-10 column or, if your amount of sample is limited, proceed to a buffer exchange using a centricon.
Good luck.
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