HELP! protein-protein cross-linking

Nikola Wenta via methods%40net.bio.net (by Nikola.Wenta from nottingham.ac.uk)
Thu Aug 7 17:32:41 EST 2008


Hi!
Maybe you can try crosslinking with glutaraldehyde. Do a time course
over 1 hour with 400 ul Protein solution by adding 8 ul 1%(v/v)
glutaraldehyde solution at room temperature. For each time point, take
out 80 ul sample, add 8 ul 1 M Tris-HCl (pH 6.8) to terminate, add 24 ul
6x SDS sample buffer and load on a gel. Depending on the initial protein
concentration, you may consider silver staining; gradient gel; are you
sure that your protein should dimerize?? How can you know? What's the Kd
value? That should be your initial protein concentration.
Cheers,
Niko


Hello, AllWe have a problem in the chemical cross-linking experiment for
protein-protein interaction. We have solved a protein complex structure,
and we want to confirm their interaction by cross-linking. But i am a
new comer in this field, the initial test was failed. Protein A is 35kd,
Protein B is 14kd, but after cross-linking the band representing 49kd is
very very weak in our SDS-PAGE comparing with the inact Protein A and
Protein B in thesame lane. The condition we used is : add DSS to protein
sample containing protein A and protein B and incubated for 1 hour under
4 degree, and then terminated it with 1M tris for 30 min. The samples
were loaded on SDS-PAGE. We have tried different concentrations of DSS
(0.1mM-10mM) and protein sample (20uM-1mM). PS: from the complex
structure , we have found several Arg residues in both protein were
located in the interface, so we use the DSS for initial test.Protein A
trends to form dimer, but we could not even got the dimer bands af!
 ter cross-linking. I would like to hear any suggestion from you.Thanks!




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