Beta-actin, double bands - Problems with my western blot (Feb/27/2007 )

Hi guys,
I hope somone can help me with this. I'm doing a standard, semi dry Western Blot using beta actin as a marker, but every time I am seeing a double banding pattern. One band (the strongest) is at the point I'd expect to see Beta-actin, and the other is about 30kD and quite weak.
Anyone got any idea as I've just been told that my last months results (over which I have slaved and was quite pleased with) are "crap".

I'm starting to loose the will to carry on!

Thanks
Rosie

-Rosie-

QUOTE (Rosie @ Feb 27 2007, 04:52 PM)

p30 may be a degradation product of actin, or a polypeptide which cross reacts with your first, (more rarely with the 2nd) Ab; check it with a different anti-actin Ab;

-The Bearer-

QUOTE (The Bearer @ Feb 27 2007, 05:52 PM)

p30 may be a degradation product of actin, or a polypeptide which cross reacts with your first, (more rarely with the 2nd) Ab; check it with a different anti-actin Ab;

I was wondering about degradation, I'm checking it out with protein extracted using a different method, I now appear to have a huge smear at 37-35kd and the smaller band at about 25kd on the membrane I probed today.
The gel is clean with no background, so my blocking is looking OK (I go for 1h in 5% milk). I'm also wondering about my ab dilution, currently I use 1:30000 fot the primary and 1:5000 for the secondary.

Anyone got any other thoughts, or had a similar problem?

Rosie

-Rosie-

Hi Rosie,

I'm having a similar problem like yours where I obtained more than one band using beta actin as housekeeping. The extra band is situated a bit lower than the expected band. Previously I tried to use GAPDH but again, I get 2 bands instead of one..

It has been months since you posted your problem here. May I know how is your blot turns out now? Have you figured out the problem? Thank you in advance..

money

-money-