Quantification of western blot using imageJ for beginners | western blot quantification | imagej |

For quantification of IHC images using imageJ, please watch this video. • Quantification of Immu...
This video lecture describes how to quantify western blot using imagej.
1. How to upload western blot image in imagej
2. How to invert image in imagej
3. How to adjust background/ brightness and contrast of western blot image in imagej
4. How to rotate image in imagej to make the lanes in the straight line
5. How to put rectangles in the image to quantify
6. How to plot lanes in imagej
7. How to make the calculation for western blot after the quantification by calculating ratios
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[@howtoneuro5930]

I wish I had this explanation a long time ago! Thank you for sharing this knowledge. Doing experiments is cool, but analyzing data is always more tricky!

[@BiologyLectures]

You're very welcome!

[@antarasengupta8614]

Thank you! It was very clear and systematic-easy to understand and follow.

[@BiologyLectures]

You're very welcome!

[@SM-xn9bv]

Excellent job!!! Very clear and consize! Many thanks!

[@BiologyLectures]

You are most welcome 🤗

[@Hari788]

Excellent explanation sir. This is what I needed

[@BiologyLectures]

Glad it helped

[@AnnieNaeem]

Thank you so much for making it easy to understand.

[@BiologyLectures]

You are most welcome. Please subscribe our channel to support us.

[@jagdeepsingh1515]

Thank you for such a beautifully explained video.

[@BiologyLectures]

Glad it was helpful!

[@dikshyapanthi7681]

Excellent explanation sir . Thank you very much for your video

[@BiologyLectures]

You are most welcome

[@kobbyblaqboi4243]

Final ratio was rather Protein r/ actin r I think. Good video! thank you.

[@BiologyLectures]

Thank you very much for your comment. In our video also, we divide protein of interest by actin.

[@ItsDeeable]

Sorry, just need a clarification - do you divide Actin Ratio by Protein Ratio or vice versa? You wrote down Actin R/Protein R but do otherwise. Thanks!

[@mallitkim]

Protein/Actin it is :)

[@huseyinkocakusak]

Very good explanation. Thank you!

[@BiologyLectures]

Glad it was helpful!

[@mrblackmamba117]

Life saver. Thank you so much sir. Subscribed.

[@BiologyLectures]

Thank you for your kind words. Thank you very much for the subscription.

[@markrobinson9676]

thank you for saving my life. awesome.

[@BiologyLectures]

Happy to help!

[@mohammedalsaegh8399]

So informative...thank you

[@BiologyLectures]

Glad it was helpful!

[@shwetab8790]

Excellent explanation. Thanks a lot.

[@BiologyLectures]

You are welcome!

[@livinghope8039]

Thanx for responding my question! I subscrbed, liked + shared your video

[@BiologyLectures]

Thank you very much for the support.

[@user-gl4zx1yd4d]

Thank you! Helped a lot

[@BiologyLectures]

You are most welcome

[@job506]

Many thanks, once again. Please, how do I obtain the relative intensity (Protein expression) for samples if the control does not have any protein expression?

[@jackbilotto4698]

Hi thanks for the video. If my control protein and protein of interest are in seperate images will the contrast settings affect the result?

[@BiologyLectures]

If you have an equal amount of samples loaded in the same order in both images, contrast settings won't affect. But if the samples are different and loaded in a different order, it will affect. Ideally, it is better to have loading control and protein of interest in the same image. We won't recommend having loading control and protein of interest in the different pictures.

[@vanesamattera2505]

Thank you for the video, how do you tag the Y axis in the plot?. Is it ok to label it like: Protein/actin (relative to control)?.

[@BiologyLectures]

Yes you can label like the way you mentioned. Or you may label like Relative band intensity (protein/actin) normalized to control.

[@mvbs824]

Thank you for the clear explanation! I had one question, what if my CTRL value is 0, how do I now calculate the ratio's and perform the normalisation?

[@BiologyLectures]

Could you please elaborate further ? Are you talking about loading control or negative control ?

[@mvbs824]

@@BiologyLectures A negative CTRL, so I have a treatment that upregulates a specific protein, and in my CTRL, there is no band visible for this protein, where there is a drastic increaase as a result of the treatment.

[@aynalsaba7982]

Thank you so much for sharing your knowledge with us. I really appreciate your work. Sir, i have one query. You have taken 1 experimental control for explaining the analysis process. Sir, i have done western blot on clinical samples and i have taken 3 control samples and 3 patient samples for my western blot process. So the thing is how will i take the ratio for those 3 controls?

[@BiologyLectures]

You can calculate the ratio for three controls and take the average to show the comparison between your control samples and experimental samples.

[@aynalsaba7982]

@@BiologyLectures again thank you so much sir. I was thinking to take average area of three control samples and then calculating the ratios but now I got the answer. I'm really grateful😊

[@tzed2219]

Very helpful!!

[@BiologyLectures]

Thank you very much 😊

[@raquelhanadulset]

I did a western blot with n=4 for both control and treatment to observe the expression of a protein of interest and then normalise against housekeeping gene .. how do I plot the data in this case? Do I calculate the ratios for each n individually? how do I carry out a paired student t-test with ratios o does it need to be with the means of the area under the curve calculated with image j? I would appreciate your help. thanks a lot!!

[@BiologyLectures]

Please calculate the ratios for each of your control and sample as described in the video. So, you will have four ratios each for sample and control. Then just put these values in excel and do the paired student t test or you can also perform the test in graphpad. Links below Excel: kzbin.info/www/bejne/Z4qlfoWMlNFlb5Y Graphpad: kzbin.info/www/bejne/lZrPZXyffL6qf5Y

[@sanja5377]

Thank you!

[@BiologyLectures]

You are most welcome.

[@roxanne3956]

At 4:22 you said there was no need for you to adjust the lanes. What I get are lanes that "don't touch the zero" so two bands have the same curve. How do I separate them?

[@BiologyLectures]

To seperate the lanes please draw the line and make it touch the zero.

[@job506]

Many thanks! I observed that some faint bands were entirely eliminated due to the adjustments of brightness/contract; how can you account for the bands. What are they, please?

[@BiologyLectures]

If you want to include faint bands also, then I would suggest to do less brightness and contrast adjustment in a way that they are still there and incorporate those in analysis.

[@job506]

@@BiologyLectures Thank you. I just wanted to be sure that they are bands of expressed proteins.

[@BiologyLectures]

@@job506 You are most welcome.

[@haleyluu9941]

What if I only am blotting for 1 protein? How would I analyze this data? I did a WB on A549 cells, probing for beta-actin.

[@BiologyLectures]

Even if you are detecting only one protein, you can use the same method to quantify. perform the calulation for beta actin only. During calculation, normalize against your control samples just as shown in the video.

[@donkeyally]

If we quantify western blot like this, our Y-axis should label relative expression, right?

[@BiologyLectures]

Yes, we can label Y axis as relative expression

[@nihanverimli2108]

Thank you

[@BiologyLectures]

You are most welcome 🤗

[@JustFelicia]

hi so if i do it like this, i do not need to subtract the background?

[@BiologyLectures]

Yes you don't need to.

[@ingridtamura1128]

I can't drag the rectangles just like you did. I tried so many times already

[@BiologyLectures]

If you are using laptop, please press and hold the left on mouse and drag it.

[@abhang4623]

any reffrence to this technique, pls provide as early as possible

[@BiologyLectures]

In this paper, we utilized this quantification approach. pubmed.ncbi.nlm.nih.gov/34453962/

[@abhang4623]

@@BiologyLectures thanks for this early response, greatful for this..

[@BiologyLectures]

@@abhang4623 You are most welcome.

[@rayanehedna6228]

I don't think it's a good thing to play on intensities like that with contrast

[@BiologyLectures]

As long as you are playing with both the control and your protein of interest, it is fine.