Hi great video I really appreciate it. I see that you have used the value of the percentage to do the normalization. Just wondering if the numbers that are left to it which were indicated by the "Area" have any use in analysis?
Thank you so much for your kind words. I am delighted to hear that you found the tutorial useful. By using the Percentage areas, it enables us to compare two or more unequal things by expressing both as portions of 100. My apology, currently, I have no idea if the "area" itself has some application for western blot quantification other than helping to calulating the percent area.
Hello, thanks for the question. In that case first the image needs to be converted into 8 bit. For background subtraction a dark background is required with white bands. So at this step it needs to be inverted. once background subtraction is performed the image needs to be inverted again to have a white background with black bands for further analysis. if the western blot bands do not require background subtraction then please proceed directly to analyze, gel, select first lane after the 8 bit image conversion.
Hello, I think ImageJ can be definitely used to perform the normalization. In this case each individual lanes (entirely from top to the bottom) needs to be selected both for the single protein and total protein expression and then perform the analysis. All the best
Hi, you are welcome. Background removal has to be uniform throughout and not only of a single band. To get an accurate quantification, selecting and removing the background especially at the selection area around the band is critical. in this video, the selected area (near the first band) for background removal would remove all the mean pixel intensities (background) uniformly from the entire blot.
Thank you for this. If I had three biological replicates on each gel and repeated the gel 3 times, how do I deal with the values please? Any idea if it is possible to use these percentages then to do a t-test? Thank you.
Hello, thanks for reaching out. Please use the normalized values to do the t test. Since you used three biological replicates on each gel and did the experiment three times, you have nine data points for each condition (eg control versus treated). After quantifying and normalizing with the housekeeping gene, choose the normalized values. Now combine the values collected from each biological replicate under the identical experimental conditions. This will result in a total of nine data points for each condition, indicating variability within and between gels. The t test can now be performed using these values.
@@nrttaye4033 Thanks a lot. I think I should take the average of every three points coming from the same biological repeat in each gel as these are considered technical repeats. What do you think? Thank you.
You are welcome. Both methods of analysis are entirely correct (average vs individual). Individual data points may provide better p values than the average of the triplicates, if the differences in the averages are substantial.
Hello, thanks for watching my video. Don't worry about it! Asking questions is how we learn and grow. I'm here to help you. Click on any region of the image outside the rectangular box .
@@nrttaye4033 Thank you very much for replying so quick and answering! Your video was so helpful too. Can I also ask, how do you deselect the box once you have already selected it as a first lane, second lane e.t.c.
@@needfulanonymity_5457 select the rectangular box by clicking on the number inside and press delete. for example in this video at 3:16 mins the rectangular selection is bounding the number 1. click on 1 and the rectangle should be selected and then it can be deleted. (During this process the rectangle selection tool should be active/selected in the ImageJ toolbar).
Hello. If the GAPDH blot is in a separate image, repeat the process you used for the gene of interest. Once results for the gene of interests are acquired, you can close the analysis entirely and begin separately for the GAPDH.
THANK YOU SO MUCH!
You're welcome! Glad you found it useful. Stay tuned for more interesting videos like this.
Hi great video I really appreciate it. I see that you have used the value of the percentage to do the normalization. Just wondering if the numbers that are left to it which were indicated by the "Area" have any use in analysis?
Thank you so much for your kind words. I am delighted to hear that you found the tutorial useful. By using the Percentage areas, it enables us to compare two or more unequal things by expressing both as portions of 100. My apology, currently, I have no idea if the "area" itself has some application for western blot quantification other than helping to calulating the percent area.
@@nrttaye4033 Thank you!!
What do you do if you are taking images of a western blot membrane that is already inverted? (white background with black bands)
Hello, thanks for the question. In that case first the image needs to be converted into 8 bit. For background subtraction a dark background is required with white bands. So at this step it needs to be inverted. once background subtraction is performed the image needs to be inverted again to have a white background with black bands for further analysis. if the western blot bands do not require background subtraction then please proceed directly to analyze, gel, select first lane after the 8 bit image conversion.
can i use this same normalization method for total protein stain?
Hello, I think ImageJ can be definitely used to perform the normalization. In this case each individual lanes (entirely from top to the bottom) needs to be selected both for the single protein and total protein expression and then perform the analysis. All the best
Hello. Thank you for the video. How to remove the background? Do I have to remove the background from one band only?
Hi, you are welcome. Background removal has to be uniform throughout and not only of a single band. To get an accurate quantification, selecting and removing the background especially at the selection area around the band is critical. in this video, the selected area (near the first band) for background removal would remove all the mean pixel intensities (background) uniformly from the entire blot.
Thank you for this. If I had three biological replicates on each gel and repeated the gel 3 times, how do I deal with the values please? Any idea if it is possible to use these percentages then to do a t-test? Thank you.
Hello, thanks for reaching out. Please use the normalized values to do the t test.
Since you used three biological replicates on each gel and did the experiment three times, you have nine data points for each condition (eg control versus treated). After quantifying and normalizing with the housekeeping gene, choose the normalized values.
Now combine the values collected from each biological replicate under the identical experimental conditions. This will result in a total of nine data points for each condition, indicating variability within and between gels. The t test can now be performed using these values.
@@nrttaye4033 Thanks a lot. I think I should take the average of every three points coming from the same biological repeat in each gel as these are considered technical repeats. What do you think? Thank you.
You are welcome. Both methods of analysis are entirely correct (average vs individual). Individual data points may provide better p values than the average of the triplicates, if the differences in the averages are substantial.
Sorry dumb question, but how do you deselect the rectangular box?
Hello, thanks for watching my video. Don't worry about it! Asking questions is how we learn and grow. I'm here to help you. Click on any region of the image outside the rectangular box .
@@nrttaye4033 Thank you very much for replying so quick and answering! Your video was so helpful too. Can I also ask, how do you deselect the box once you have already selected it as a first lane, second lane e.t.c.
@@needfulanonymity_5457 select the rectangular box by clicking on the number inside and press delete. for example in this video at 3:16 mins the rectangular selection is bounding the number 1. click on 1 and the rectangle should be selected and then it can be deleted. (During this process the rectangle selection tool should be active/selected in the ImageJ toolbar).
Nice
Hello, thank you. glad you liked the video
May I ask how to normalize the protein band with GAPDH loaded in another gel?
Hello. If the GAPDH blot is in a separate image, repeat the process you used for the gene of interest. Once results for the gene of interests are acquired, you can close the analysis entirely and begin separately for the GAPDH.
@@nrttaye4033 Thank you very much!
you are welcome