Hi
I am trying to find a method to measure the total protein content in my western blots. I am thinking of using the SYPRO Ruby dye. However, I am not sure if there is a software or a method to quantify the bands that fluoresce after the staining. Does anyone have experience with that?
Thanks
Sushmita
Hi Sushmita,
The software I would recommend is ImageJ (or more specifically Fiji, which is ImageJ with a number of science addons). You can download it here: https://fiji.sc/
Plenty of people have used ImageJ to analyze Western bands, and so as long as you have a standard to use (where the concentration is known), it's just a matter of following these steps:
https://lukemiller.org/index.php/2010/11/analyzing-gels-and-western-blots-with-image-j/
Hope that helps, and good luck!
I assume you're already using a loading control?
If you want to use a dye, you can try Coomassie or Ponceau just to get the total protein in the gel.
Licor has a lot of information on this topic. Checkout:-
http://www.licor.com/bio/blog/western-blotting-2/total-protein-stain-as-an-internal-loading-control/
We are currently trialing their stain REVERT. Ponceau is very insensitive. I think the Licor solution may be expensive.
You can also use stain free gels
https://www.youtube.com/watch?v=J-d1iKIozKw
Depends, Coomassie would be the simplest solution if you are not expecting low quantitites of proteins. InstantBlue goes down to 5ng
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