Dye for protein affinity measurement

From: Jiang Jiahong
Date: 8 February 2012 13:43

 Dear all,
I am looking for some kind of dye for protein affinity comparison, but do not know which to choose.

I know  protein A can contact B to form a complex,now I hope to find something simiar with A to act as an inhibitor to block the process of A-B complex formation. Maybe a short peptide, a segment of protein A or even some organic molecule.

Because here is a poor access to ITC nor Biacore, I can only rely on some dye to check the competence between A and inhibitor candidates.

If any one can offer any suggestions. That would be so grateful! Any way,thank kind-hearted people in advance!

    Regards
     Jiahong

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From: Patrick Shaw Stewart

Jiahong

Thermo sells a series of kits called DyLight Fluor for fluorescent labelling of antibodies or other proteins.  They have everything you need and they're very convenient and easy to use.  You can pick the excitation and emission wavelength.  If you label both A and B (or C) with different "colors" you will be able to see if both are in your crystals (assuming crystallization is part of your approach).

You need only label a small percentage of your protein or peptide to see whether the protein is present in a crystal.

Patrick

http://en.wikipedia.org/wiki/DyLight_Fluor

Forsythe, E.L., Achari, A., and Pusey, Marc L. (2006),  Trace Fluorescent 

Labeling for High Throughput Crystallography, Acta Cryst. D62, 339-346. 

We used DyLight 350 NHS Ester to check we had protein crystals - see 

methods section of Cryst. Growth Des., 2011, 11 (8), pp 3432–3441

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From: Xiaodi Yu

Hello Jiahong:

If I understand correctly that you want to test protein-protein interaction or inhibition study in solution, maybe you can try something like ELISA to test protein-protein interaction. Or if your B protein has 6 histag, you can use Ni-NTA agrose beads to test inhibition or binding depending on your purpose. And another option (a little "dangerous" ), is using radio active to label one of your protein.

Yu Xiaodi

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From: Vandana Kukshal
Hi,

•       If your protein is making strong complex then You can run Native page  with increasing concentration of your inhibitor peptide and decrease in complex band intensity will show you competitive binding of your inhibitor to  proteins. 
•  You can do ELISA... by coating one of your protein ad then add second protein and then detect by using antibodies against second protein ... with increasing concentration of peptide or inhibitor signal should go down. 
•  if interaction is weak then design a peptide (part of protein B ) which definitely binds to protein with Alexa , or FITC labelled and do interaction study by observing the change in fluorescence. and if interaction is there do assay with different inhibitors ... 

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Vandana kukshal