Berglund:Calculating protein concentrations

From OpenWetWare
Jump to: navigation, search

I found this calculator to work the best for me:

http://molbiol.ru/eng/scripts/01_04.html

Take note, and this is very important. The method chosen for quantitating you protein must be chosen very carefully.

See for an understanding of how the Bradford can vary from protein to protein:
labs.fhcrc.org/fero/Protocols/BioRad_Bradford.pdf

As a lab we have tested BCA, bradford, UV, qubit.

The best method that I have for now (Amy) is:

Do a bradford assay using the Biorad reagents. Compare these results against a serial dilution on a SDS-PAGE gel stained with Coomassie (or something like Sypro Ruby fluorescent protein stain) and compare against serial dilutions of BSA or IgG. The bradford method is much more sensitive to IgG than to BSA and IgG is the preferred protein to use for your standards (www.applichem.com/fileadmin/produktinfo/a6932_de.pdf)

It's particularly important to compare against the gel if your protein is degrading or if you have contaminant proteins.

Here is a method I use for the bradford:
http://www.science.smith.edu/departments/Biochem/Biochem_353/Bradford.html

Amy