Loading control Issues and Normalization

[br.gruenewald]

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

[LedBulb900Lumen]

i don't

[obama]

Originally Posted by br.gruenewald

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.