Hi, - If ur 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 ...
On Wed, Feb 8, 2012 at 8:13 PM, Xiaodi Yu <[email protected]> wrote: > 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 > > ------------------------------ > Date: Wed, 8 Feb 2012 14:17:47 +0000 > From: [email protected] > Subject: Re: [ccp4bb] Dye for protein affinity measurement > To: [email protected] > > > 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 > > > > > 2012/2/8 Jiang Jiahong <[email protected]> > > 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 > > > > > > > > -- > [email protected] Douglas Instruments Ltd. > Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK > Directors: Peter Baldock, Patrick Shaw Stewart > > http://www.douglas.co.uk > Tel: 44 (0) 148-864-9090 US toll-free 1-877-225-2034 > Regd. England 2177994, VAT Reg. GB 480 7371 36 > > -- Vandana kukshal
