Kinase Assay - lots of bands? (Sep/19/2008 )
Hi Everyone,
I tried out my first ever kinase assay yesterday. I added P32 labeled ATP and a GST-tagged substrate to my IP. I ended up with lots (really, lots) of bands after exposing it on film overnight. I don't have a positive or negative control (tisk, tisk), but I have a feeling this is not what it's supposed to look like. Any input on what I should see and suggestions for my next attempt?
Thanks!
-montana-
QUOTE (montana @ Sep 19 2008, 08:34 AM)
Hi Everyone,
I tried out my first ever kinase assay yesterday. I added P32 labeled ATP and a GST-tagged substrate to my IP. I ended up with lots (really, lots) of bands after exposing it on film overnight. I don't have a positive or negative control (tisk, tisk), but I have a feeling this is not what it's supposed to look like. Any input on what I should see and suggestions for my next attempt?
Thanks!
I tried out my first ever kinase assay yesterday. I added P32 labeled ATP and a GST-tagged substrate to my IP. I ended up with lots (really, lots) of bands after exposing it on film overnight. I don't have a positive or negative control (tisk, tisk), but I have a feeling this is not what it's supposed to look like. Any input on what I should see and suggestions for my next attempt?
Thanks!
you co-immunoprecipitate various other substrates and kinases which are complexed to your substrate or the antigen; beware that labeling with radioactive ATP is very sensitive;
-The Bearer-
I must be pulling down lots of other kinases that are phosphorylating lots of other stuff (?). I think my IP antibody is not so good. I will try a different one as suggested by a lab member and see how it goes. Thanks for the input!
-montana-