How to determine band intensity of Western blot? - High School Student in need of help for the Science Fair! (Feb/03/2005 )
I am doing an experiment using the Western Blot for my science fair project.
I heard that in order to produce a graph, the intensity of bands must be evaluated using adobe photoshop 5.5 or 7.0. I was wondering if someone can tell me please, step by step how to go about in obtaining the pixels/intensity of the bands generated using the program?
Thank you! I appreciate the help!
I heard that this can be done with photoshop, but i have no first-hand experience in that. in our lab we use ScionImage from Scion Corp.
You can get this here, though support is only given to paying customers...
but then, maybe someone comes around who uses PS for quantification and can help you out...
I'm not gonna tell you the procedurebut i want to point out about the quality of quantitation. honnestly he single method i've heard consists in loading on the SAME GEL your extracts and a know quantity of proteins 2, 4, 6, 8, etc... with different proteins of different molecular weight and if you use antibodies, to reveal you film with the same exposure time (in practical that says a corevelation). If you want just an aproximate (but not so bad) value you can use photoshop as said by jadefalcon.
First thing you have to do is get a GOOD exposure of your gel. This means you DON'T overexpose. Rule of thumb is you have to be able to see through the bands (i.e. put it up against a piece of paper and see if you can read soem text through it..)
Secondly, ALL the bands you want to compare have to on the same film. The quantification is all relative towards one band you put as 1 (or 100%).
1) In PS, load you your image
2) Then do, Image>Adjust>Invert (this way, you invert the scale)
3) Draw a box, which covers ONE band using the Marquee Tool (do the biggest band first, as the box HAS to be the same size all way through the analysis. This way to don't have to take the box size into consideration)
4) Then do, Image>Histogram and write down the Mean
5) Then drag the box to the next band and repeat
6) Finally, drag the box to measure the Mean from the Background, and subtract the number from all the others.
7) Divide all the numbers with your reference number for the one set as 1 (or 100%)
This is a VERY rough way of doing it, but do give you some hints on the relative increase/decrease of expression.
Just remember when you make a digital image of your film using a scanner, you need to create the highest quality image possible. We scan our films in grayscale and produce a 1200 dpi resolution TIFF file.
Do not make a jpeg of your film, jpegs contain far less image data then tiff files.
Id try out using the Scion image above. I dont have any experience using photoshop to quantify the bands as well.
Load the tiff file into Scion image. Ive never used scion image, but ithe pprinciple i suppose would be the same in any image program. Im sure this method is far from perfect, but It might work for you
First draw a box in an area that has no bands and select analyze->measure. This will tell you the background intensity of your image (mean), and also the area of your box Write these numbers down.
Next, draw a ROI (region of interest) around a band that you want to measure and select analyze-> measure. Again youll get an area measurement, and a mean intensity measurement. Write these numbers down. Repeat this for all the bands you want to measure.
Use care in making your ROIs and I'd experiment drawing ROIs and comparing the values you get from them until you think you can draw them properly and get an accurate measurement.
I might be wrong, but in order to quantify your bands, youll need to multiply the mean intensity and the area together to get the number you want. Do this also for your background. Write these numbers down.
Next subtract the background value (area x mean intensity) from each of your band values (area x mean intensity)
LEt me know if this makes sense. Also if anyone can improve my described method, dont hesitate to do so
I'm not sure how to quantitate the intensity of the bands, but there's free software called ImageJ (from the NIH) that is downloadable from the internet that our lab uses to do densitometry analysis of our Western blots.
Why not provide an exact address.
please note the date of that last post
I suspect you could find the address if you used google? or, alternatively, searched the NIH site?
Another one on western blot quantification and Scion Image (or ImageJ)…
Here is an illustration of the results I get:
With results like the ones illustrated on the image, wouldn’t it be better to take into account the size (area) of the band and not only its intensity ???
I have very little experience using Scion Image but, when using the Gelplot Macro, I can only select the different lanes with the rectangular selection tool and the size of the rectangular selection has to stay the same for each lane.
Instead of drawing area of interest, I would like to use the magic wand selection, but this implies thresholding the picture, is it a good idea ?
Do you apply any kind of modification (threshold/enhance contrast/…) on the image prior to the measurements ?
Instead of subtracting a single background value for the whole film (from a rectangular selection taken anywhere on the film), is it a good idea or a waste of time to subtract a background value to each lane (taken just above or just under the corresponding lane) ???
@ snolan6: thank you for the step by step method !