Someone that work with human primary cells and western blot: how many cells I need to identify protein abundance in western blot analysis?
I want to quantify Caspase-1 and gasdermin D
In my experience, you should get ~200-300 ug protein from 1 million cells.
You can quantify protein with Bradford assay, and use ECL reagent for Western. Hope you also have a good antibody against your protein.
Good luck!
1 million cells will enough, it is recommended to run an SDS-PAGE in advance
Enrico Castroflorio Have you ever tried with 200k of cells? Is it enough to detect a protein in WB?
The absolute threshold of detection (e.g. pmol, nmol, etc...) for a protein depends largely on the properties of the primary antibody and the method being used to image the blot. It depends on how well the primary antibody binds to the protein in question at a concentration that doesn't produce undue non-specific, secondary bands.
With regards to detecting a protein in a cell lysate, it will further depend on how abundant that protein is in the type of cell being analyzed. As a result, the absolute number of cells needed to reliably detect a protein depends on the expression profile of that protein.
Most Western Blots that I have seen published use somewhere between 5 and 50 ug total protein. If your antibody is any good at all, 20 ug should be more than enough protein for detection if it's expressed at an appreciable level. As others have said, 1 million cells and even 200k cells should contain more than enough protein to load this amount.
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