quantify bands - (Jul/22/2005 )

hello everybody,
i have to do a lot of western blots in this period, but i have no experience with this technique: what kind of software is the best to quantify my bands? (i have to calculate the percentage of knockdown of a gene)
thanks
Mauro

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Lots of free programs that are reliable, try ImageJ Gel analyzer

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Hello!

I've got a related question regarding quaititating bands. We use GeneQuant (Biorad) and the software requires one to draw a rectangle around the band of interest. The software then measures band intensity and subtracts the background from this value to give the adjusted intensity. The issue that i have is that the bands are often not exactly the same size. So if I want to cover the entire band, the rectangles are not going to be the same size... I was told that the area of the rectangles have to be the same in order for the quantitation to be relative. But if that's the case, then the rectangles for the bigger bands will overlap...if the rectangle is smaller, then the larger bands don't get covered...so i'm worried that not all of the intensity is being accounted for.

Has anyone encountered this problem? and how do you do it? Pls help! I am reluctant to analyze results without clarifying this. Thanks in advance.

ggUss

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hi
a recent discussion may help
http://www.protocol-online.org/forums/inde...=quantification

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First let me say that yes your boxes must be the same size and if you have to overlap then do it.

Second,
I have had a similar problem, and I would suggest using a different method available in the quantityone software. What you do is open your gel then you make lanes with the lane command--the line should be in the center of the lane.. then you do a detect bands command. you can adjust the background for each lane and look at the peaks (peaks are like scion image software) that represent the intesity at that position in the lane using the lane background command. I use this peak to determine optimal background. then you do the all lanes report command and use the int*mm2 (area under the curve) for your quantitation. If i have not explained clearly enough and you want to try--repost and I will respond as soon as I can....

hope that this helps...

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