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-   -   Can't get loading controlto work after stripping (http://www.molecularstation.com/forum/western-blot-forum/3815-cant-get-loading-controlto-work-after-stripping.html)

psingh 08-24-2008 09:01 PM

Can't get loading controlto work after stripping
 
My antibodies work beautifully and I get a nice difference in protein expression between tumor and control samples. My problem is, after I strip the membrane using harsh stripping protocol (@ Abcam using beta-mercaptoethanol) and wash thoroughly for 30 min under water and probe with beta-acting, I get no signal.

My PI has in the past, made it work and I don't know why it doesn't work for me. Can any one help me out.

I have re-used the primary Ab only once before and I was very very careful. Is there a possibility I over-washed the membrane?

If anyone can give me any insight on what to do next or what you think went wrong, it will be very helpful.

psingh 08-24-2008 09:04 PM

Re: Can't get loading controlto work after stripping
 
What do people usually do to check for loading control? Does everyone strip the membrane or there is a way around this? someone suggested cutting the membrane if there the protein bands are of different sizes( in my case,my protein is 17kD and beta-actin is 40kD), but my PI is against this (I may try it in his absence, but is this advisable?)

moleculardude 08-24-2008 10:04 PM

Re: Can't get loading controlto work after stripping
 
hey there,
this may be due to several reasons such as different membrane, more sensitive antibody, or different stripping buffer composition / stripping method.

If you are wasting time and money, try investing if your PI can afford it into a more gentle stripping buffer if may save a lot of time and money.

If you cant do this, I think you should either do 2 things:

1) Dual primary antibody incubation with your protein of interest and the positive control (you have to be careful your antibodies dont crossreact and that the proteins are of different size etc)

or:

2) Optimize your stripping to be less harsh - make sure also your proteins are staying on your membrane by using a good membrane.

Let us know how you go

moleculardude 08-24-2008 10:04 PM

Re: Can't get loading controlto work after stripping
 
hey there,
this may be due to several reasons such as different membrane, more sensitive antibody, or different stripping buffer composition / stripping method.

If you are wasting time and money, try investing if your PI can afford it into a more gentle stripping buffer if may save a lot of time and money.

If you cant do this, I think you should either do 2 things:

1) Dual primary antibody incubation with your protein of interest and the positive control (you have to be careful your antibodies dont crossreact and that the proteins are of different size etc)

or:

2) Optimize your stripping to be less harsh - make sure also your proteins are staying on your membrane by using a good membrane.

Let us know how you go

psingh 08-25-2008 09:55 PM

Re: Can't get loading controlto work after stripping
 
My proteins are of sizes 17kD and 77kD and beta-acting is around 40kD. Is it not cutting it too close especially 17 and 40?

Also, how do I make sure that my anti-bodies do not cross react?

Is there a mild stripping protocol you recommend? Is it more expensive? I have been referring to Abcam for protocols as that is what my PI does and it seems cheap.

I figured that I RE-USED the beta-acting anti-body dilution from the last time my PI had made it work. Is it possible that re-using the house-keeping gene may be the problem as it would have been completely used? Has anyone ever come across this problem and can give me an insight if they think this may be the reason for faliure.

psingh 08-25-2008 09:56 PM

Re: Can't get loading controlto work after stripping
 
sorry for my mis-spelling of beta-actin which has been acting wierd for me.

admin 08-25-2008 10:07 PM

Re: Can't get loading controlto work after stripping
 
Hi Psingh,
yes reusing (freeze thaw) can affect many antibodies. Where was the antibody stored? How long?

I would try it again with fresh antibody and use the same stripping protocol your PI used, it may work without buying more expensive stripping buffers etc.

Let us know how it goes.

psingh 08-26-2008 09:38 PM

Re: Can't get loading controlto work after stripping
 
the antibody was stored at 4 degrees for a week at a dilution of 1:1000. is that re-usable for beta actin?

admin 08-26-2008 10:14 PM

Re: Can't get loading controlto work after stripping
 
Hello Psingh, that may have been the issue as we usually would store our reused antibodies at -20. Have you tried the experiment again with fresh antibody?

psingh 08-27-2008 11:19 PM

Re: Can't get loading controlto work after stripping
 
I stripped the membrane and reprobe with fresh beta-actin. Now all I can see is the ladder. There is no signal from the other protein lanes. Any idea what is going on? Also, I barely see the ladder after exposure of 30 min (membrane to film).


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