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!
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!
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?
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 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😊
@@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.
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?
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?
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.
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!!
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: ru-vid.com/video/%D0%B2%D0%B8%D0%B4%D0%B5%D0%BE-1YBMRV_l294.html Graphpad: ru-vid.com/video/%D0%B2%D0%B8%D0%B4%D0%B5%D0%BE-_il4IiGYwI4.html
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.
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.
