Western Blotting for Phospho-protein

dave2006 · #1 (**permalink**) 02-07-2006, 03:00 AM

Hi,
I am trying to western Blot for Phospho-protein. Right now I am getting no signal. What can I do?

thanks in advance

liono · #2 (**permalink**) 02-09-2006, 03:32 AM

Hey dude,

1) you should lyse in a buffer with phosphatase inhibitors ie Vanadate

2) you should keep everything cold

3) you should use the lysate immediately

4) when blotting you should usually incubate overnite in the cold room 4 degrees C

5) lyse in small volumes of SDS-buffer and load everything

hope it helps
liono

dave2006 · #3 (**permalink**) 02-12-2006, 06:51 PM

thanks for the great reply, i am redoing the blot the way you mentioned
dave

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Western Blotting for Phospho-protein

Signalling Biology

Signalling is an integral part of the cell neural network. Phosphorylation and other protein modifications modulate this interaction. Post and discuss the study of protein-protein interactions, and signalling biology protocols and science here.