Quantifying bands on SDS-PAGE using ImageJ

[@Faisal.Alqhtani]

Thank you so much; you have helped me a lot.

[@RahulPatharkar]

You are welcome!

[@suckmemore]

for years I have seen ppl use "Analyse --> gel --> plot lane tool" method for quantification. I think I like this one better!

[@RahulPatharkar]

Thanks!

[@MonaraKaelle]

Thank you very much for sharing that. It helps us a lot. I have some questions: - why dont you use Analyse --> gel --> plot lane tool? - if I understood well, each fraction represents one sample with different proteins run, including urease. In your case, you compare urease:lane ratio among fractions. Whether the brightness of lanes changes, the proportion of urease changes as well. So, do you want to analyse how much urease changes according to other proteins in the fraction? Or do you want to compare only urease quantities among fractions without interference of other bands brightness (other proteins)? - Why don't you normalize by reference band (actin, GAPDH...)? Again, thank you! :-)

[@RahulPatharkar]

I not sure I follow. How would plotting the entire lane help us get the intensity of a single band?

[@MonaraKaelle]

@@RahulPatharkar I guess that I didn't understand neither hahah. You do a relative signal of urease to the rest of the lane. In this case, what is the the goal of such measurement? I don't understand very well because urease as well the other proteins in the lane can vary among them.

[@RahulPatharkar]

@@MonaraKaelle The goal is to quantify the amount and relative purity of the urease enzyme after gel purification steps. I hope this helps.

[@MonaraKaelle]

@@RahulPatharkar Yes, It did. Thank you very much. I suggest put that on description of the video.

[@murakamidarwish76]

Can I use a SDS's picture from my smartphone camera to apply this method? Or I need to take a picture of SDS from specific machine?

[@RahulPatharkar]

A carefully taken image from your smartphone can work well.

[@EAli-ec1xp]

Thank you very much. Can this method apply on western blot anlysis??

[@RahulPatharkar]

Yes, you can apply this method for Western blots.

[@tessiaw.3196]

Thank you so much for the video, it really helped me! :)

[@RahulPatharkar]

You are very welcome. All the best, Rahul

[@vu6812]

Great video! Is there a way I can measure the background? I try to subtract the background to get a better purity % for my protein. I selected the well it was empty and measured it. But the area number was almost as high as with my protein well. Do you have any suggestions? Thanks

[@RahulPatharkar]

Yes you can and there are a number of ways to do it. One way would be to simply measure and appropriate area of your gel image that you think could be the background with imageJ just as you measure bands. Then in Excel subtract that value from your actual bands. I hope this helps.

[@paulabulieris7515]

Very helpful.

[@RahulPatharkar]

Thank you

[@thiagooliveira4035]

Thanks for the video. What do I do when the IntDen value exits the same as RawIntDen?

[@RahulPatharkar]

Thiago, that will happen when no "Set Scale" (in Analyse Menu) is set. Many images from scanners will have a scale associated with them, like 300 pixels/inch. If this is not specified, IntDen and RawIntDen will be the same. You can manually set the scale by going to "Set Scale". For relative quantifications it is not necessary to set the scale. I hope this helps.

[@karlabarron1454]

¡Hello good day! I liked the video, but I have a question. In case the image is from PCR products, is the same procedure done to quantify?

[@RahulPatharkar]

Yes, you can do exactly the same thing for PCR products on agarose gels. Thanks and all the best.

[@vodka7280]

thank you for the video. I have a question, if i have a few gels for measurements and comparison, how do i standardise the baseline intensity for all of the bands in these gels?

[@RahulPatharkar]

It is best practice standardize the baseline for each gel separately because there are often small differences in staining background and image capture for each gel. To get a baseline, draw a measurement box on a blank area of the gel that has the same size as the box you would put over the band (move the box with the cursor keys so that the box is identical). Subtract the background measurement from the band measurement.

[@ivnazg1]

is it necessary to invert the photo so the background is black and bands are white? What is the difference between IOD and optical density calculated by plotting the peaks and mark then manually?

[@RahulPatharkar]

Inverting the image makes bands have positive signal values (black = zero). For your second question, please explain further what you are asking about "plotting the peaks and mark then manually".

[@ivnazg1]

@@RahulPatharkar In available videos on KZbin covering this topic, bands are marked with the rectangle tool, like in this video, but then with functions analyze - gels - plot lanes, peaks are plotted which represent the signal (density) of specific band. In this video, density is obtained simply with measure function, without plotting peaks.

[@aritin5259]

Hi there! How could I apply this to a TLC, multicolored sample? I really only need to quantify the “white” areas so should I measure the white areas individually? Or is there a way to black out everything else but the white areas? Thanks for the great video!

[@RahulPatharkar]

You should be able to use exactly the same principles for TLC band quantification. Convert you image to black and white then invert your image if necessary so that your bands are white. Just watch all of the steps in the video and I think your problem will likely be doable. I hope this helps.

[@kxhcjxbg205]

Hi Which software is best and why image j or gel analyzer for anlyzing gel i want quick response i m very worried can anyone help me plz in this difficult time.

[@RahulPatharkar]

Image J is free and it works.

[@kxhcjxbg205]

@@RahulPatharkar Another reason??

[@RahulPatharkar]

@@kxhcjxbg205 All gel image analysis programs do the same thing so free is an important reason. Also, image J has more plugins for scientific image analysis than pretty much any other program.

[@diyana4933]

are those bands in lane 1-6 are purified bands?

[@RahulPatharkar]

Lanes 1-6 are fractions from a gel filtration column. The bands are only partially purified.

[@diyana4933]

@@RahulPatharkar ok thank you very much!

[@sasha8181]

What does integrative density measure?

[@RahulPatharkar]

Basically it is measuring the intensity of the image feature you have selected which in this example is band intensity. I hope this helps.