western to detect ATM - (Dec/13/2006 )
Hi!!
Just wanted to check out whether anybody out here has done a successful western blot for ATM (ataxia telangiectasia mutated). I have a protocol as of now; but, I'm not very sure if that's going to work for ATM since, the protocol I have is a general one and ATM is a slightly different protein since it's big in size. Any suggestions or tips would be really helpful and appreciated!:)
Thanks in advance!!
-swati-
As ATM is a large protein you should omit Methanol from your transfer buffer. You will also get much better resolution if you run your Western on a Tris-Acetate or Tris-Bis gel - you can get premade ones from Invitrogen - I would recommend the 3-8% Tris-Acetate. The antibody I have used is from Gentex, use at 1:1000.
Good luck!
-Mawdryn-
QUOTE (Mawdryn @ Dec 22 2006, 07:50 AM)
As ATM is a large protein you should omit Methanol from your transfer buffer. You will also get much better resolution if you run your Western on a Tris-Acetate or Tris-Bis gel - you can get premade ones from Invitrogen - I would recommend the 3-8% Tris-Acetate. The antibody I have used is from Gentex, use at 1:1000.
Good luck!
Good luck!
Why tris-acetate Gel is better than Tris -HCl gel? Is it possible that we could do a good western on ATM with Tris-HCl gel system (such as the 4-12% gradiant gel from bio-rad)?
-flakie-
QUOTE (Mawdryn @ Dec 22 2006, 08:50 AM)
As ATM is a large protein you should omit Methanol from your transfer buffer. You will also get much better resolution if you run your Western on a Tris-Acetate or Tris-Bis gel - you can get premade ones from Invitrogen - I would recommend the 3-8% Tris-Acetate. The antibody I have used is from Gentex, use at 1:1000.
Good luck!
Good luck!
Thanks a lot for your suggestions. Can I ask you one more favor? I'm looking for the recipe of : 3% Tris-acetate gel, running buffer and transfer buffer.
I'm trying hard to find the same. If by chance you have the recipe do you mind sharing it?
Your help would be greatly appreciated:)
-swati-