I need to detect and measure the abundance of cytoplasmatic proteins (CASP1 an GSDMD) by western blot in human mo-DCs, and some companies where I purchased the antibodies do not specify a antibody concentration to the assay.
How I can test the best concentration?
Which minimum and maximum antibody concentration I should test to definy the best concentration?
In my opinion, 1:1000 would be an optimal concentration for this Abs. However, for testing an optimal conc. I would propose to test a concentration range of 1:100 to 1:2000 (e.g. 1:100, 1:250; 1:500; 1:1000; 1:2000).
If you don't know the specific working concentration of your antibody preparation, you can start with following concentration according to the source of antibody.
Tissue culture supernatant (1/100), Ascites (1/1000), Whole antiserum (1/500), Purified antibody (1ug/mL).
You can optimize by adjusting concentration first for getting your expected data.
In my opinion, 1:1000 would be an optimal concentration for this Abs. However, for testing an optimal conc. I would propose to test a concentration range of 1:100 to 1:2000 (e.g. 1:100, 1:250; 1:500; 1:1000; 1:2000).
- Agreed with the Above
- These things are ab/Epitope specific but generally 1:100 to 1:200 is not atypical
- What I would say However is that antibodies that are validated for Western blotting will come with guidance on the data sheet supplied by antibody purveyor
- If that information isn't available then as mentioned try 1: 100 to 1: 200 but also google your antibody and look for prior studies where these factors have been published in 'materials and methods'
Dear Amanda, your first choice should be the information leaflet comes with the antibody you intend to use for the westerns, which has definite recommended concentrations to use for the western blot and elsewhere.
The quality of antibody determines its concentration to be used for different applications and this is judged by their manufacturing scheme like are they monoclonal, polyclonal, cross-adsorbed, carrier-free or even crude. Also for loading control (housekeeping) antibodies concentrations can be quite less as compared to your target ones.
The following site is very useful and highly recommended one to check about your antibody and its citations and concentrations used for western blot or in other immunoassays. It is really a splendid site. Have a look please and it will solve all of your problems related to any antibody in the future too.
https://www.citeab.com/
Regards....
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