[Protein-analysis] Re: HELP! protein-protein cross-linking

jps via proteins%40net.bio.net (by sumida from bbri.org)
Mon Aug 11 18:27:16 EST 2008


I suppose some intial questions would have to do with some basic
issues which I'm sure you have been careful about but one needs to ask
them anyway in order to move forward.
1)  Is your buffer free of reactive amines ie. presumably it is not a
tris or imidazole type buffer?
2)  Is your reagent fresh?
3) Are you working in a pH range 7-9? And are you sure?

Second order concerns would have to do with the site accessibility of
DSS to the reactive amines on the protein.
Do you know if this site could be sterically blocked?
How does trypsin interact with your protein?  Chances are if trypsin
does cleave at the reactive site or if the rate of cleavage at the
reactive site is slowed, there are steric issues with the accessibity
of the probe to the reactive site.
Do you know if there are charged groups that could result in a
coulombic interaction with your probe?

Of course these latter more interesting queastions should only be
addressed after the less interesting but more important questions are
satisfactorily answered.

=B0=FC=C8=F1 wrote:
> From: baorui_ren from hotmail.comTo: methods from net.bio.netSubject: HELP! protein=
-protein cross-linkingDate: Tue, 5 Aug 2008 02:28:20 +0000
>
>
> Hello, AllWe have a problem in the chemical cross-linking experiment for =
protein-protein interaction. We have solved a protein complex structure, an=
d we want to confirm their interaction by cross-linking. But i am a new com=
er 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 w=
eak in our SDS-PAGE comparing with the inact Protein A and Protein B in the=
same 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 t=
erminated 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 samp=
le (20uM-1mM). PS: from the complex structure , we have found several Arg r=
esidues in both protein were located in the interface, so we use the DSS fo=
r initial test.Protein A trends to form dimer, but we could not even got th=
e dimer bands after cross-linking. I would like to hear any suggestion from=
 you.Thanks!


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