Dephosphorylation of protein in tissue lysate - going insane,please help! (Jan/19/2006 )

Hi there,

Please please please help. Ive been trying to do this simple experiment for weeks now, someone must have done it!
I am enquiring about dephosphorylation of PROTEIN in a TISSUE lysate. I am trying to find out if a protein of interest is phosphorylated in a certain diseased state. So far I have tried dephosphorylation using alkaline phosphatase CIP, but it has not dephosphorylated any phospho proteins. Can you suggest an alternative product/protocol for me to use? Note that I cannot IP my protein due to tiny sample volumes, so it must be done on crude lysate. No cell line or tissue have these phosphorylated forms.

Thank you in advance for your help,

[quote name='rose1980' date='Jan 19 2006, 11:40 AM' post='37604']
Hi there,

Please please please help. Ive been trying to do this simple experiment for weeks now, someone must have done it!
I am enquiring about dephosphorylation of PROTEIN in a TISSUE lysate. I am trying to find out if a protein of interest is phosphorylated in a certain diseased state. So far I have tried dephosphorylation using alkaline phosphatase CIP, but it has not dephosphorylated any phospho proteins. Can you suggest an alternative product/protocol for me to use? Note that I cannot IP my protein due to tiny sample volumes, so it must be done on crude lysate. No cell line or tissue have these phosphorylated forms.

Thank you in advance for your help,


Hi!

Mu suggestions are: to go to this link:http://www.neb.com/nebecomm/products/protocol18.asp

also, check the Ph in your lysate_ must be optimal for CIP or any other enzyme youve been using,

FINALLY, take the SAP_ it's also not bad for the proteins.

Regards,