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!
@BiologyLectures2 жыл бұрын
You're very welcome!
@antarasengupta86146 ай бұрын
Thank you! It was very clear and systematic-easy to understand and follow.
@BiologyLectures6 ай бұрын
You're very welcome!
@kobbyblaqboi42432 жыл бұрын
Final ratio was rather Protein r/ actin r I think. Good video! thank you.
@BiologyLectures2 жыл бұрын
Thank you very much for your comment. In our video also, we divide protein of interest by actin.
@ItsDeeable2 жыл бұрын
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!
@mallitkim9 ай бұрын
Protein/Actin it is :)
@SM-xn9bv11 ай бұрын
Excellent job!!! Very clear and consize! Many thanks!
@BiologyLectures11 ай бұрын
You are most welcome 🤗
@dikshyapanthi76812 жыл бұрын
Excellent explanation sir . Thank you very much for your video
@BiologyLectures2 жыл бұрын
You are most welcome
@job5065 ай бұрын
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?
@AnnieNaeem2 жыл бұрын
Thank you so much for making it easy to understand.
@BiologyLectures2 жыл бұрын
You are most welcome. Please subscribe our channel to support us.
@markrobinson9676 Жыл бұрын
thank you for saving my life. awesome.
@BiologyLectures Жыл бұрын
Happy to help!
@aynalsaba798211 ай бұрын
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?
@BiologyLectures11 ай бұрын
You can calculate the ratio for three controls and take the average to show the comparison between your control samples and experimental samples.
@aynalsaba798211 ай бұрын
@@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😊
@shwetab8790 Жыл бұрын
Excellent explanation. Thanks a lot.
@BiologyLectures Жыл бұрын
You are welcome!
@mvbs824 Жыл бұрын
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 Жыл бұрын
Could you please elaborate further ? Are you talking about loading control or negative control ?
@mvbs824 Жыл бұрын
@@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.
@livinghope8039 Жыл бұрын
Thanx for responding my question! I subscrbed, liked + shared your video
@BiologyLectures Жыл бұрын
Thank you very much for the support.
@Hari7882 жыл бұрын
Excellent explanation sir. This is what I needed
@BiologyLectures2 жыл бұрын
Glad it helped
@user-gl4zx1yd4d7 ай бұрын
Thank you! Helped a lot
@BiologyLectures7 ай бұрын
You are most welcome
@jackbilotto4698 Жыл бұрын
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 Жыл бұрын
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.
@raquelhanadulset2 жыл бұрын
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!!
@BiologyLectures2 жыл бұрын
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: kzfaq.info/get/bejne/Z79ygLWHxNKWaWg.html Graphpad: kzfaq.info/get/bejne/lc-cZ6yarL_beWg.html
@jagdeepsingh15152 жыл бұрын
Thank you for such a beautifully explained video.
@BiologyLectures2 жыл бұрын
Glad it was helpful!
@ingridtamura11282 жыл бұрын
I can't drag the rectangles just like you did. I tried so many times already
@BiologyLectures2 жыл бұрын
If you are using laptop, please press and hold the left on mouse and drag it.
@huseyinkocakusak2 жыл бұрын
Very good explanation. Thank you!
@BiologyLectures2 жыл бұрын
Glad it was helpful!
@job506 Жыл бұрын
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 Жыл бұрын
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 Жыл бұрын
@@BiologyLectures Thank you. I just wanted to be sure that they are bands of expressed proteins.
@BiologyLectures Жыл бұрын
@@job506 You are most welcome.
@roxanne39562 жыл бұрын
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?
@BiologyLectures2 жыл бұрын
To seperate the lanes please draw the line and make it touch the zero.
@vanesamattera2505 Жыл бұрын
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 Жыл бұрын
Yes you can label like the way you mentioned. Or you may label like Relative band intensity (protein/actin) normalized to control.
@mrblackmamba1172 жыл бұрын
Life saver. Thank you so much sir. Subscribed.
@BiologyLectures2 жыл бұрын
Thank you for your kind words. Thank you very much for the subscription.
@tzed22198 ай бұрын
Very helpful!!
@BiologyLectures8 ай бұрын
Thank you very much 😊
@donkeyally2 жыл бұрын
If we quantify western blot like this, our Y-axis should label relative expression, right?
@BiologyLectures2 жыл бұрын
Yes, we can label Y axis as relative expression
@JustFelicia Жыл бұрын
hi so if i do it like this, i do not need to subtract the background?
@BiologyLectures Жыл бұрын
Yes you don't need to.
@haleyluu99412 жыл бұрын
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.
@BiologyLectures2 жыл бұрын
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.
@rayanehedna6228 Жыл бұрын
I don't think it's a good thing to play on intensities like that with contrast
@BiologyLectures Жыл бұрын
As long as you are playing with both the control and your protein of interest, it is fine.
@mohammedalsaegh83992 жыл бұрын
So informative...thank you
@BiologyLectures2 жыл бұрын
Glad it was helpful!
@sanja5377 Жыл бұрын
Thank you!
@BiologyLectures Жыл бұрын
You are most welcome.
@nihanverimli2108 Жыл бұрын
Thank you
@BiologyLectures Жыл бұрын
You are most welcome 🤗
@abhang46232 жыл бұрын
any reffrence to this technique, pls provide as early as possible
@BiologyLectures2 жыл бұрын
In this paper, we utilized this quantification approach. pubmed.ncbi.nlm.nih.gov/34453962/
@abhang46232 жыл бұрын
@@BiologyLectures thanks for this early response, greatful for this..