Hi all,
I trying to cross link between a membrane protein ~170kDa and Chaperones. I have some problems due to mega complexes and problem to lysis the sample.
I am using 2mM of DSP stoping the reaction with 10mM Tris pH7.4.
it is not working correctly.
Any one ever worked with DSP?
Guy

I've used DSP and other similar linkers in the past. What specifically did you mean when you said it was not working correctly?

Also, what is your sample and why did you choose DSS?

After running the lysis I cant see the band of one of my proteins of interest.
It seems that I have problem in lysing after the corss link was stoped. (protocol as described by the company).
Guy

Regarding to this DSP cross linker.
What kind of reverse method to break the link would you use.
Do you think that 10 bME is good. I am afraid of heating my sample.
Guy

OK,
I found the best solution to cleave the cross linking.
100mM DTT in Sample Buffer, Incubte for 25min at 37deg.
Guy

Hi again,
Anyone had some problems when trying to lyse after the incubation of 0.2mM DSP?
The lysis efficiancy drops as the DSP conc. goes up.

Important !!!
From several experiments that I have done it seems that DSP effects degrdation of my protein. Maybe it dose it also to other protein which I guess is the case.
Guy