Hello, I want to perform Bradford assay to quantify my protein samples. The assay is not compatible with my buffer I have used RIPA which contain detergent. I wanted to use acetone to precipitate my proteins and remove the detergent. My question is what is the best methode to remove detergent with acetone and after precipitation how to elute the protein which what buffer to recover a good amount of my proteins.
You can use a different assay to quantify proteins. Thermo says that BCA is compatible with RIPA.
In conclusion you want to good protocol for protein isolation followed by quantification. So, you need to invest some time to play with the parameters in the isolation method for your specific sample types. Some times its not easy to adopt any method as it is. There are lot of changes and alternatives required for the successful protocol.
Hi. It is true that Bradford gives higher absorbance with detergents and buffers like RIPA, NP40 etc. You may try using BCA method. However, if you are quantifying proteins to determine the loading amount in PAGE, you may very well use Bradford only where you do not need an accurate quantification as such. Having said that, you may use BCA using the available kits (from BioRad and other companies) to avoid any error.
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