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We used RIPA lysis buffer and wondered whether it will have an effect on results. The RIPA buffer includes 150mM NaCl, 0.1% Triton X-100, 0.1% SDS, 0.5% sodium deoxycholate, and 50mM Tris-HCl. Another question that we have is that we used the Bradford Assay to determine our protein lysate concentrations. Do you think the protein concentrations determined from the Bradford is accurate enough, i.e. is there a better method that you would recommend to determine protein concentrations?
Asked on Nov 18 2013
The ELISA assays are compatible with RIPA buffer. However, you should prepare your standards and positive controls in the same buffer.
The Bradford assay is compatible with RIPA buffer if the samples are diluted at least 10-fold with distilled water. Thermo Scientific/Pierce has a useful guide to protein assays in PDF form at the following link. http://www.piercenet.com/files/1602063_PAssayHB_122910.pdf . On page 12-13 you will see a table that compares the compatibility of various protein assays with many reagents, including RIPA buffer. The table refers to the Bradford assay as "Coomassie", an alternative name. The BCA assay is considered to be a better choice than the Bradford assay for assays of samples that contain detergents, but if you dilute your samples before assaying them, the Bradford may be adequate.
Tom RuyleAbcam Scientific Support
Answered on Nov 18 2013