Anti-phosphothreonine in ELISA (KIRA assay)

Kevin Moulder Kevin_Moulder at mail.biogen.com
Mon Jun 17 11:29:17 EST 1996

We are attempting to configure a KIRA (Kinase receptor activation) assay which 
detects phosphorylation of cell-surface proteins by ELISA. We are using 
anti-phosphothreonine (from Sigma) and are running into trouble with background 
- which appears to be caused by adding cell-lysates to plates coated with 
monoclonal antibody (to capture the receptor of interest) and blocked with 
either bovine serum or gelatin.

Could phosphorylated Fc receptors be binding to antibody on the plate and then 
be detected by anti-phosphothreonine??

Do blocking proteins contain phosphoproteins???

Has anyone attempted a similar assay and encountered the same problems.

Thanks  Kevin Moulder 

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