Hi Sanjeev,
In addition to all the excellent suggestions (trying different buffers/salts,
co-expression, concentrator, incubate and set up crystallization screening,
checking SPR, fusion), I have a couple more suggestions:
1) related to your question about cross-linker: do you have
] For stabilizing protein-protein complex
Not observing a complex on a gel filtration run for a week complex (micromolar)
is not necessarily unexpected. It all depends on your protein concentrations,
the dissociation kinetics (remember, a gel filtration run is not an equilibrium
experiment
Not observing a complex on a gel filtration run for a week complex
(micromolar) is not necessarily unexpected. It all depends on your
protein concentrations, the dissociation kinetics (remember, a gel
filtration run is not an equilibrium experiment - it dilutes and
separates your sample), etc.
If your goal is a crystal structure, this reference may be worth consulting.
The paper describes the use of centrifugal concentrators to make a complex of
two proteins that have Kd of 19 micromolar for crystallization. They mixed the
two proteins and then concentrated using a membrane that
n 2017 21:35:32 +0530 (IST)
Subject: [ccp4bb] For stabilizing protein-protein complex
Dear all,
I am trying to stabilize a protein-protein complex. Our SPR study indicates
it is having micro molar dissociation constant. I tried to purify both the
molecule in complex form with size exclusion chromato
C.UK
> Enviadas: Terça-feira, 31 de Janeiro de 2017 14:05:32
> Assunto: [ccp4bb] For stabilizing protein-protein complex
> Dear all,
> I am trying to stabilize a protein-protein complex. Our SPR study
> indicates it is having micro molar dissociation constant. I tried to
> purify bot
are kon and
koff—fast I assume?
JPK
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Ursula
Schulze-Gahmen
Sent: Tuesday, January 31, 2017 11:30 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] For stabilizing protein-protein complex
We have had good luck with creating fusion
We have had good luck with creating fusion proteins of the 2 proteins in
question (http://www.nature.com/articles/ncomms14076). If you don't know
how they interact, you would need to try different linker length and
different order of the 2 proteins in the fusion protein. It would also be
helpful
Dear all,
I am trying to stabilize a protein-protein complex. Our SPR study indicates
it is having micro molar dissociation constant. I tried to purify both the
molecule in complex form with size exclusion chromatography (mixed both the
protein in equal molar ratio and incubated at 4 degree for 1
