This video was made by Maryville College Biology student Lauren Evans as part of her Senior Study. It shows the procedure for quantifying protein bands using ImageJ software.
Hi, thank you for the video. I can't get to measure the areas. When I use the straight line tool, the line is yellow and not blue. And when I do a second line, the first one disappears. So I can't measure all the areas in one time. What sould I do?
After I select my first area and click "set first lane", I drag my second box to the next band, which is fine. Each time then go to "select next lane", the selection automatically moves back to the first region, labelled with two. I can then not remove both overlapping selections, without staring again. Any suggestions as to how to avoid this would be appreciated!
I have a problem when doing this. The lanes will only be vertically placed and not horizontal. It is as if ImageJ "see" the gel as if it was flipped 90 degrees. when i rotate the picture 90 degrees. the lanes can only be placed horizontal and not vertically? it is so weird??
Its dimensionless, you can tell that it´s your "signal", or call "units". It´s probably the integrated area below the curve, which maybe is the number of pixels darker than your background. If you want to convert this data into something useful, you should add one or more standards (curve) in known concentrations, in such way you plot their signal x concentration. Or you can divide the signal of one band by the sum of the signals of all bands in that well, so you can find the purity.
Very helpful. But I have a problem. My interest protein has 2 isoforms, so for each lane I have 2 bands, and the plot gave 2 different peak. How do I measure them? each area of each band separate and then calculate the mean?
Hi Mathlouthi, Thanks for your question. You can press the Ctrl key on the first band and drag the yellow box to next band, now you can do Ctrl+2. When you want to draw next band you can follow the same step that I mentioned. I think its works. Thank you
This is likely super late, but for other people asking the same question: That is just a signal. You need to change it to concentration by making a plot with your standards
@@book_worm88 you basically test different known concentrations in your gel (called standards), and use their signal to plot a line (x=concentration; y=signal). Then you can use this plot to get the concentration of any signal within your limit of detection. Remember to get the y=mx+b formula from your curve (Excel can give it to you automatically). Hope this helps/ make sense.
Or you can divide that band´s signal by the sum of the signals of all bands in that well, so you can find the purity. In this case, I prefer the GelQuantNET software.
thanks! turns out you can explain shit in 3 minutes and don't need a 45 minute lecture to make you feel like a "fookin expert" scientist. I wish more people in the field realized that.
Thank you very much for making easy but extremely useful video!
Thank you for the detailed introduction!
That was such an amazing video! Thanks!
Thank you so much. I got it . This video is very useful and explained very easily as well.
Thanks for sharing. What should I do if I have background for the bands?
Thanks a lot for sharing! This is helpful!
Very practical and useful!
Than you so much, very informative video. Good luck.
Hi, thank you for the video. I can't get to measure the areas. When I use the straight line tool, the line is yellow and not blue. And when I do a second line, the first one disappears. So I can't measure all the areas in one time.
What sould I do?
Very helpful, thank you very much!
thank you very much for teaching me how to read gels
Thank you!
Thanks a lot!!
Congratulations for your video so useful ;)
THANKS A LOT FOR THAT!!!
Thank you. Very useful*****
Great video- thanks
How can we compute then protein quantities ?
After I select my first area and click "set first lane", I drag my second box to the next band, which is fine. Each time then go to "select next lane", the selection automatically moves back to the first region, labelled with two. I can then not remove both overlapping selections, without staring again. Any suggestions as to how to avoid this would be appreciated!
use arrow on keyboard to move box, it´s working, I had the same problem before
+Šárka Zweitakt I have the same problem when i use the arrows on my Keyboard. It always switches back to the forst region.
Could you solve this? I have the same problem..
I had this problem too. How to solve it? Thanks.
Try to change to 8 bit your image before to do the lane and repeat the process
I have a problem when doing this. The lanes will only be vertically placed and not horizontal. It is as if ImageJ "see" the gel as if it was flipped 90 degrees. when i rotate the picture 90 degrees. the lanes can only be placed horizontal and not vertically? it is so weird??
Thanks a lot ! It is so easy :p
very elaborative and helpful.. thanks.. would be highly obliged if you can help me out with the calibration curve preparation and quantification
thank you so much!
Thanks, great.
what are the units of the final numbers you got?
I am also interested!
Its dimensionless, you can tell that it´s your "signal", or call "units". It´s probably the integrated area below the curve, which maybe is the number of pixels darker than your background. If you want to convert this data into something useful, you should add one or more standards (curve) in known concentrations, in such way you plot their signal x concentration. Or you can divide the signal of one band by the sum of the signals of all bands in that well, so you can find the purity.
Very helpful. But I have a problem. My interest protein has 2 isoforms, so for each lane I have 2 bands, and the plot gave 2 different peak. How do I measure them? each area of each band separate and then calculate the mean?
how to convert area into the concentration of protein? i still dont get it
thanks a lot
Thank you for the tutorial. But when I select "Select Next Lane" it dosen't work for me... does anyone has an idea how to solve this?
Thank you
Hi Mathlouthi, Thanks for your question. You can press the Ctrl key on the first band and drag the yellow box to next band, now you can do Ctrl+2. When you want to draw next band you can follow the same step that I mentioned. I think its works. Thank you
thank u,,
Saved me a lot of time and headache
Mam my image is an RGB color. Is it ok or should I convert it into a grayscale image? Please tell me ASAP
thank u
What are the units of the area????
pixels I believe
A.U.
Super stupid question, but when you get the result, there is 4629,782 of... what? what is the unit?
This is likely super late, but for other people asking the same question: That is just a signal. You need to change it to concentration by making a plot with your standards
@@peaceman5044 how can I do that? would you please help
@@book_worm88 you basically test different known concentrations in your gel (called standards), and use their signal to plot a line (x=concentration; y=signal). Then you can use this plot to get the concentration of any signal within your limit of detection. Remember to get the y=mx+b formula from your curve (Excel can give it to you automatically). Hope this helps/ make sense.
Or you can divide that band´s signal by the sum of the signals of all bands in that well, so you can find the purity. In this case, I prefer the GelQuantNET software.
@@peaceman5044 Thanks!
It will be wonderful if we can measure DNA peaks
You can.
thanks! turns out you can explain shit in 3 minutes and don't need a 45 minute lecture to make you feel like a "fookin expert" scientist. I wish more people in the field realized that.