protein degredation - (Jun/19/2008 )
I am working with the protein which is 16kd. My protein is becoming degraded when I am checking my western blot result and I don’t get the good band.
I run my SDS page gel for 1hr and western blot for 1 hr too.
Do you have any suggestion?
when you say degrade, are you implying that you have good band before that and now you get a weak band? You can always use any of the protease inhibitor cocktail.
Did you use Ponceau Red after western blot to check the transfer? I usually run 2 hours for western blot though. good luck!
Your protein really shouldn't degrade much once you have it in the SDS gel loading buffer, so if you're having degradation problems then it's most likely a prior step. Add protease inhibitors as suggested, also PMSF as well.
Acutally there are many reasons indeer.
We can separate into three main parts:
1. Protein Preparation.
2. SDS Separation.
3. Western Blotting.
If you really want to find out the problems, you need to solve it one by one.
At first, protein preparation is very important factor to determine whether you sample is good or bad for running the gel and Western. After protein preparation, you should better to do the protein analysis to check the concentration of protein in you sample.
2. For running the SDS, u can try to load the max. concentratino of protein sample in the gel to get the maximum signal.
3. After running the SDS, u can do the Western Bloting. U need to check the antibody . Also after Western, you can use the gel to do the silver-stain, you can still find the protein .
Try to solve the problem by your own@@Work hard