Two bands in the attached blot are very close to each other, and I am unable to quantify separately.
Is it fine to quantify together and take an average of that?
The bands are not non specific .
Thank you.
Hi Rohini,
You could a free software called image J. http://rsb.info.nih.gov/ij/
This will allow you to highlight the 2 bands and give you 2 peaks. You can then measure the density of each band and have results for your band of interest.
If you needmore help let me know.
Hi Rohini,
you could try to use a lower percentage for you SDS-PAGE to get a better separation of you bands. Since you don't have any information on the percentage I can just suggest this point in general.
If it is fine to quantify both together? Since you didn't tell anything about the band or the purpose of your quantification approach it's not possible to give you a good advice on that. Are these phosphorylated and unphosphorlated-variants of your proteins? Isoforms?
Some more information would be very useful for others to give you a helping hand.
Best
Kai
It depends on the base you are taking. If these bands a part of the total 100% reaction, include them. If you ignore them, don't forget to describe this.
Hi Rohini,
You could a free software called image J. http://rsb.info.nih.gov/ij/
This will allow you to highlight the 2 bands and give you 2 peaks. You can then measure the density of each band and have results for your band of interest.
If you needmore help let me know.
I agree with Afthab. Use the software. Different bands usually mean different proteins (or homologs) and trying to quantify them as one is not the right thing to do to obtain individual protein expression.
The software will help you.
Thanks Afthab,
Can you please explain how to analyse the result from the image j software.
In the results section i got area (same for all bands) and the mean value.
Should i take the mean value as the result.
Thank you,
Rohini
Dear Rohini,
The software i recommend is called image tool and not image J.
to download use this link http://compdent.uthscsa.edu/dig/download.html .
Run the program and then open your image using this software.
select the box tool and draw a box over your 2 bands and then press the number 1.
Then select the box you have just drawn and move over the the next column, when its in the correct place press the number no 2 on your keyboard, then select the box again from box 1 and move the the third column and PRESS No 2 again.
When you have finished boxing all your columns press no 3 on your keyboard and a new sheet opens up with peaks. select the line tool drawing from 1 peak to the next. select the wand icon and click anywhere inside that peak and the software will tell you the density of the first peak that represents the density for the first band etc.
good luck.
Hi Rohini,
Unfortunately I do not recommend the densitometry of these bands. Since your bands are not "non-specific" it is not scientifically correct to take an average of them together. A average will not represent the intensity of each band.
The best choice would be to repeat the race.
Could you try to perform a blot with an higher acrylamide concentration?
than try to use 12 or 15% of acrylamide solution. I am sure that the two bands will be spaced. good luke
Hi Rohini,
My suggestion would be try to resolve it not from image analysis but improving protein separation using more quantity of acrylamide in your gel. This could provide you better bands.
Good luck!
Hi Rohni,
Keep in mind that if you do not properly separate your two bands and quantify them together, you will be flattening your signal modulation if you are seeking a regulation. If the non-relevant band is not modulated, you would be underestimating your regulation if the two bands overlap a bit. You seem to be saying the two bands are specific signal. If you mean they are actually two isoforms of your protein of interest, then you should be able to measure them together and check for a modulation of the overall signal. Be careful how your interpret the measurement afterwards. You might need to check for the regulation using a different method. If you need to further separate your bands, you can use a tricin-based gel instead of a polyacrylamide gel.
Jean-Charles
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