Hi ,
yes as *Konstantin* said if u have cysteine residue in ur any one
of protein u can label that protein with Pyrene malemide or oregon
malemide or other fluorescence dye then remove the dye by desalting column.
titrate with second protein and do fluorescence anisotropy experiment to
get the KD value (stochiometry).
one more thing u can do. u can add kinase motif in one of ur protein by
re-cloning and then label with labelled ATP and then do EMSA with other
protein.
regards
On Thu, Aug 4, 2011 at 9:13 AM, Konstantin v. Korotkov <
k...@u.washington.edu> wrote:
> Hi Min,
>
> If your proteins have cysteine residues, or you could introduce them into
> the sequence, then you could label the interacting proteins with a
> fluorescent label, run an SDS-PAGE and quantify the bands with a fluorescent
> scanner. See for example Supplementary Figure 1C in Fronzes et al. (2009)
> Science 323: 266.
>
> -Konstantin
>
>
> On Wed, 3 Aug 2011, m zhang wrote:
>
> Dear all,
>> I have two unrelated questions. Any suggestion on any of them will be
>> greatly appreciated.
>>
>> First, we have two proteins that bind each other without a doubt. But
>> since we have very limited amount of proteins and it takes a long time to
>> reproduce them, we are very hesitating to try ITC and AUC to find out the
>> stoichiometry of these complex. So I am wondering if there is any other
>> way we can try to find the ratio without consuming much proteins?
>>
>> Second, we are thinking about getting a new incubator for crystallization.
>> Could anyone here recommend a model or a brand?
>>
>> Thank you,
>>
>> Min
>>
>>
>>
>>
> --------------------------
> Konstantin Korotkov, Ph.D.
>
> Research Scientist
> University of Washington
> Department of Biochemistry
> Box 357742
> Seattle, WA 98195-7742
>
> (206)616-4512
> k...@u.washington.edu
> --------------------------
--
Vandana Kukshal
Postdoc Fellow
structure biology group
international Center For Genetic Engineering and Biotechnolgy
India
