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Loading control Issues and Normalization

Loading control Issues and Normalization - Western Blot Forum

Loading control Issues and Normalization - Discuss western blotting and immunoblot in the western blot forum discussion board. Ask Questions about transfers, blocking, membrane antibody incubation and exposures.


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Old 01-03-2012, 03:17 PM
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Default Loading control Issues and Normalization



Hi All,

I'm EXTREMELY new to doing western blot analysis, as this is the first time I've gotten to do this.

I have an issue. Vinculin has been used as the loading control for the blot, however on some of my samples, the vinculin signal is very faint. This makes normalization against it extremely difficult because when dividing the numbers from the sample by the number I get from the vinculin, I have a 4 fold increase and this isn't correct! I've searched and searched all over the web for an answer. Is it possible that I still have to divide these numbers and get the larger fold value and then is there a way to do possibly an adjusted normalization value? I'm not really sure where to go from here. I had 3 samples that had this happen on but the loading controls were fine on all my other samples.

Any advice helps!!!

Thanks in Advance!

-Brandi
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Old 01-04-2012, 09:00 AM
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Default Re: Loading control Issues and Normalization

i don't
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Old 01-04-2012, 12:52 PM
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Default Re: Loading control Issues and Normalization

Quote:
Originally Posted by br.gruenewald View Post
Hi All,

I'm EXTREMELY new to doing western blot analysis, as this is the first time I've gotten to do this.

I have an issue. Vinculin has been used as the loading control for the blot, however on some of my samples, the vinculin signal is very faint. This makes normalization against it extremely difficult because when dividing the numbers from the sample by the number I get from the vinculin, I have a 4 fold increase and this isn't correct! I've searched and searched all over the web for an answer. Is it possible that I still have to divide these numbers and get the larger fold value and then is there a way to do possibly an adjusted normalization value? I'm not really sure where to go from here. I had 3 samples that had this happen on but the loading controls were fine on all my other samples.

Any advice helps!!!

Thanks in Advance!

-Brandi
if you are not convinced by this result. then reprobe the same blot for actin and recalculate the result.
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