Hi! I have a problem with CXCR4 run. I should see the bands around 40 kDa but I see the staining at the beginning of the gel, I supposed that CXCR4 is not able to migrate. First time I tryed I boiled my samples at 100 °C for 5 minutes and I got this type of results (no migration), that I tryed to not boil the samples but put them at 70 °C for 5 minutes but I got the same problem.
I know that at high temperature CXCR4 aggregates with other proteins, but also not boiling samples I have the same problems. Does anyone has some suggestion?
Thank you!
Daniele: How did you lyse your sample containing CXCR4? Did you use 2x Laemmli's buffer containing SDS (but not b-ME)? If that is the case, you are encountering a complex of CXCR4 with other (chaperone) proteins connected with disulfide bonds. We've seen this kind of phenomenon in almost all serum proteins complexed with alpha2-macroglobulin or complement C3, and detected this as a ~720 kDa band on SDS-PAGE (almost at the top of the gel).
Hi Daniele
Have you tried DTT instead of ß MSH ? ßMSH (or b-ME) is reversible and increase, in proteins that contains cysteins, the probability to form multimers by introducing disulfide bonds between two monomers. Currently, DTT avoid this problem.
Thank you very much for your answers!
Yes, I use to add DTT (in the lisis buffer, during the extraction) and even the B-ME (before the SDS-page). Should I try to avoid B-ME in order to decrease the disulfide bonds? Thank you!
Ok! I'm going to try as soon as possibile and I will let's you know.
Thank you Mr. Nadal and Mr Zhao also.
Dear Pr. Nadal, Do you know if I have to replace DTT with B-ME with exactly the same quantity or must I to be carefull about the DTT concentration?
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