western blot - inconsistent western blot (Jan/14/2010 )

Hi! my western blot for myosin light chain 20 from a tissue is giving me trouble. onetime I get excellent, clean band. Other time I get little tiny bands obscured by high background everywhere. I use exact same primary n secondary antibodies in exactly the same dilutions. Everything is kept identical everytime, then why do I get such extremely varrying results?

Also, I notice that same amount of same protein run on two different gels gel 1 and gel 2 (both 10% SDS-PAGE gels) at the same time gives me stronger signal in one gel and weaker signal in another. Is it possible that Nitrocellulose membrane that I use could have gone wrong to cause this inconsistency? I have made sure that gel 1 and gel are exact same when I make the gels.

-Bhupal-Reno-

Have you had a good result only once? Or does it vary from time to time (one day its good, the other its bad, then it turns back good again)? Here's a couple questions that may help you solve this problem.

Could your sample have degraded?
Does your antibody tolerate freeze/thaw cycles well?
Do the washing time vary from one experiment to the other?
Could your various buffers (migration, transfert) have gone bad?
Is your transfert apparatus working consistently?
Do you have the same problems with other blots?
Have you tried PVDF for transfert instead of nitrocellulose?
Have you tried blocking with 5% milk? or 2% BSA?

For your different results from 2 identical gels :

Do you use the same antibody solution? Or do you make different ones?
Are the gels handled at the same time? Exact same times of washing, transfert, resting etc?

Obtaining identical results from 2 different gels may be tricky, but once get comfortable with WB, it gets pretty easy. But in your case, it seems to me that something is wrong. You only have to figure it out!

-madrius1-

madrius1 on Jan 14 2010, 08:15 AM said:

one day its good n other day its bad with the same sample. If sample has degraded then it should give me permanently bad blots right? Its like on and off. antibody is new. same conditions and same reagents and everything is same. Acually, aftr the transfer I cut the same membrane in three pieces and blot for three different proteins I get consistent results for other two proteins. we have been using nitrocellulose for all the proteins that we stain in the lab. We use 2% Milk protein with 0.2% tween-20 for blocking.

for 2 different gels

yes, i do same time, transfer the 2 gels in the same tank together for duplicate resulst. everything I keep same. I use same antibody.

-Bhupal-Reno-

Then, if you can blot with consitent results for other proteins, it seems that your problem is your antibody. Some ab don't behave well in milk. You shoud try blocking with 2% BSA instead of milk. If you can, I would also try PVDF membranes.

And if all of the above doesn't work, I would try to find another ab for this particular protein.

Good luck!

-madrius1-

Thank you for your sugesstions. But how can same antibody go wrong in less than a week. I had good results just a week before and now everything remaining the same, I get high background with obscured bands. I will certainly try 2% BSA as per your sugesstion though.

thanks

-Bhupal-Reno-

Bhupal-Reno on Jan 16 2010, 02:53 PM said:

Hi can anyone tell me what is the role of 0.2% tween-20 in blocking buffer and washing buffer used for immunoblotting in westerns.

-Bhupal-Reno-

Bhupal-Reno on Jan 17 2010, 11:57 PM said:

Hi can anyone tell me what is the role of 0.2% tween-20 in blocking buffer and washing buffer used for immunoblotting in westerns.

it helps reduce non-specific binding of the antibody.

-mdfenko-