Looking for CRO for Activity Profile of Candidate Non-ATP-Competitive Inhibitors of Src Kinase
We have a client who is looking for a provider (worldwide) who will be able to assist in running experiments for determining activity profiles of candidate non-ATP-competitive inhibitors.
The client has suggested a design of experiments for determining activity profiles of candidate non-ATP-competitive inhibitors:
I. Determining Kd and IC50 of candidate kinase inhibitors* of c-Src kinase
1) Express and purify two forms of tagged Src (in which Tyr416 is unphosphorylated and phosphorylated tyrosine) in cell lines (literature suggests SF9 insect cells); confirm phosphorylation states using immunoprecipitation and Western blotting
2) In tumor cell lines: determine Kd and IC50 of candidate inhibitors compounds to the two kinase forms, in presence of ATP-Mg2+. Based on literature search, the most suitable method is likely to be fluorescence spectroscopy: Lebakken et al. J Biomolec Screening 2009
II. Determining phosphorylation state of a substrate (Cas) of Src upon inhibitor binding
1) Same as 1) in part I. but express Cas kinase as well.
2) In tumor cell lines:
a) Add Src to the cell line first
b) After a period of time, add candidate inhibitor to the solution
c) After a period of time, add Cas to the solution
d) Detect whether Tyr325 on Cas gets phosphorylated using immunoprecipitation and Western blotting
3) Positive and negative controls will be included.
*Final pool of candidate inhibitors will be determined by the end of May 2015. The candidate inhibitors to be tested currently consist of ZINC compounds.
If you can conduct these assays or have alternative methods to carry out these experiments, please provide technical feedback, pricing information, and turn around times through the system so that we may pass the details onto our client to review.
Thank you in advance for your kind assistance and we look forward to hearing from you and working with your team on this project.
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