Hi everyone,
I am trying DSS crosslinking for my protein. My protein is expected to make trimer or hexamer with Ub. To prove this, I am crosslinking my protein with DSS. I tried crosslinking for 5',15',45' and 60 minutes. My protein's MW is 98 kDa with HisMBP tag. At 5', I observed some protein in well and a band corresponds to 98kDA which is monomer size of my protein. But as I continued from 15' to 45', 60 minutes , I observed faint bands to no band (at 60') corresponds to my monomer size. I tried running gel for longer time with 5% SDS PAGE gel as well. But my protein (oligomer) seems to be stuck in wells. Could you please help and suggest me solution regarding this.
I have attached gel picture for your reference. Thank you.
Did you try to run on a gel with low concentration, such as 4-6%?
Try lowering the DSS concentration and/or the protein concentration during crosslinking.
Hi,
you can make different reactions in which you can vary both the protein and DSS concentration. Besides, perform reaction in ice as the reaction is very fast at room temperature. I run 6% SDS PAGE gels to check the cross-linked products. You can also optimize the reaction time since long exposure creates non specific aggregates.
Good luck
Hi, Ritu
Try to lower the concentration of crosslinker and try to modify the incubation time. Your blot shows that there is already some crosslinking just after 5 minutes and a lot after 15 minutes.
Also, you could reduce the sample directly before running it on a gel. Since this will break up the crosslinked proteins, you will not see them anymore, but if your protein is somehow purified you can access the experimental conditions that allow you to crosslink your his-tag protein to Ub.
Good luck
Gisela
Thank you very much everyone for your kind suggestions. I'll try what is suggested.
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