ERK by Westerns - (Jun/22/2006 )
Hi all,
I am trying to detect phospho-ERK using an antibody from Abcam (rabb polyclonal ERK1 + ERK2 (phospho T202/183 + Y204/185). Without any success. Phosphorylated ERK is constitutively expressed in my fibroblasts evidently.
Conditions as follows:
Protein denatured with boiling in reducing buffer
30ug protein on 10% polyacrylamide gel blotted onto PVDF.
Blocked in 5% milk (just discovered that this is a no-no), so tried 3% BSA
1:1000 primary antibody
1:1000 secondary (anti-rabb HRP)
Developed with ECL and exposed for anywhere of upwards of 15 min.
My ladder comes up nicely, so it is not the secondary.
Anyone used the Abcam antibody before? Anyone got any hints?
Thanks in advance.
I've done a bit of phospho ERK blotting and I had to try a lot of different antibodies before I found one that didn't give strong signal all the time. I think if the antibody binds just a bit to non-phosphorylated ERK it gives the appearance that ERK is always phosphorylated. Although using fibroblasts, it is possible that this is indeed the case.
Do you serum starve the cells before giving your treatments or whatever? I found this was really key in reducing background in my untreated cells.
Also, do you always use 1:1000 for your secondary antibody? that seems like a lot too me but obviously we are using different reagents. Do you have a lot of background?
Good Luck,
Mountainman
Sorry for the confusion. I guess I wasn't clear. I get no bands on the western, except for the ladder.
Sorry I have no experience with anti phospho ERK from Abcam.
I used the one from Promega (anti-active MAPKs) followed Promega protocol, and it worked perfectly. (even with milk, once).
are you able to detect ERK with an anti-ERK antibody I mean independently of its phosphorylation?
I haven't tried ERK alone. I suspect that would be a good starting place.
I am currently running extract from fibroblasts that has been stimulated with TGF-beta. This increases ERK phosphorylation substantially.
I am currently running extract from fibroblasts that has been stimulated with TGF-beta. This increases ERK phosphorylation substantially.
I run ERK westerns all the time, I use the cell signaling Ab, its great, I also have issues with
high background phospo, and I am going to try serum starving, although I worry about the other
changes in signaling that this would lead to?!?!
Everyone seems to use the Cell Signaling antibody. Maybe I should have checked it out before I bought Abcam.
yip, the antibody is good, the only problem is my results are rubbish!!!!
Sorry to hear that