Dear all,
After protein extraction with a RIPA buffer (50 mM Tris pH 7.5, 150 mM NaCl, 1% NP-40, 1% Na-deoxycholate, 0,1% SDS, 1mM EDTA +PIC), I wanted to quantify the yieild by Bradford assay. The RIPA I used was transparent and no precipitates were visible.
When I added the RIPA to the Bradford buffer (for the blank) a weird blue precipitate was formed in the tube, the color and the apparence makes me think those are not proteins.
Do you know what could have precipitate? I used this RIPA once already and I didn't have this problem.
Do you think I could still use the extracted proteins for Mass Spectrometry?
Hello Giulia Perrotta
You could still use the extracted proteins for Mass Spectrometry. The problem is the Bradford assay. This protein assay is not compatible with detergents. SDS used in RIPA buffer used to extract proteins, interferes with Bradford assay. When proteins that carry residue of SDS are used as samples in the Bradford assay, the Coomassie dye is either kept from binding due to the bondage of SDS to proteins, or the SDS associates with the green form of the dye, shifting the equilibrium and over representing the absorption at 595 nm regardless of true protein concentration.
For more information you may want to refer to the paper attached below.
https://www.sciencedirect.com/science/article/abs/pii/0003269789906362
To make the assay somewhat compatible, dilute your protein quantitation samples at least 10-fold with water or PBS to reduce the amount of detergent and consequentially lower the interference. Or you may use the BCA protein assay. This method is compatibility with many detergents at concentrations up to 1 or 5%. You may want to refer to the link provided below for the protocol.
https://assets.thermofisher.com/TFS-Assets/LSG/Application-Notes/TR0008-TCA-acetone-precip.pdf
Best.
Hello Malcom,
Thanks for your answer. What I don't understand is why I had this problem this time. I used in the past the same RIPA recipe and it didn't react with the Bradford.
I can only think that after 3mo the buffer has to be prepared fresh...
Giulia
Hello Giulia,
I too had thought the same. Maybe you are right. The shelf life of RIPA lysis buffer is 1 month when stored at 4 deg C.
Best.
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