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
hi Min,
I think SPR(Surface plasmon resonance) should suit your situation. The
Biacore instrument I worked on required 200 µL of 10-50 µG protein(ligate)
for immobilisation and 150µL of various concentration of the other
protein(ligand) in the range of 50-1000 µg/ml. (depending on your situation
Dear Min,
You can use microscale thermophoresis for this kind of tricky cases when you
have limited amount of protein and ligands. There is enough literature on
this topic.
have a look in the following reference:
Proc Natl Acad Sci U S A. 2006 Dec 26;103(52):19678-82
Hope this helps.
Regards,
Hi Min,
Given the limited supply of your protein you could try the EDC-NHS zero-length
crosslinking either in one-step or two-step protocol (Anal. Biochem.
185,131,1990). If your protein complex is like most protein complexes chances
are there will be some electrostatic contacts at the
Dear all, Thank you all very much for your kind suggestions. Now we have some
preliminary data of SPR. But we might try other new ideas suggested here.
Best,Min
Date: Thu, 4 Aug 2011 13:19:41 +0200
From: krishna@gmail.com
Subject: Re: [ccp4bb] stoichiometry of complex and incubator
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
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.
