Hi for all. Because Bradford precipitates with detergents of RIPA buffer, can I prepare standards in dionized water and finally subtract the readings of my unknown samples from the readings of the buffer alone?
You can try DC (Detergent compatible) or RC DC Protein Assay kit from BioRad.
http://www.bio-rad.com/en-us/product/rc-dc-protein-assay
Hi Yassar, as Arvind suggested, you might want to switch to a protein assay that is not affected by detergents, such as the one from BioRad, or BCA from Pierce:
http://www.piercenet.com/product/bca-protein-assay
It will be much easier to switch to an assay that works with detergents rather than try to modify the Bradford assay. It will require a lot of work to convince people that your modifications are working.
If you are planning to publish your results, it is much easier to use an accepted protein assay that works in the presence of detergents rather than explaining how you got a protein assay to work that should not work under these condition.
Hi,
I am sorry to tell you that you cannot use protein standards for the Bradford assay unless they contain all the components that are present in your protein samples. That is the main reason why do we have so many different protein assay kits on the market, some are more expensive than the others. It is recommendable that you use detergent-compatible assay kit, as suggested by others. Or, try to search through the literature, if you find some articles that succeded in the application of the Bradford in the presence of detergents then cite them. Be careful with the removal of detergent before the protein assay, as your proteins may aggregate and that is not compatible with any protein assay. I use BCA assay for membrane proteins in the presence of Triton X-100.
Hi Yassar
If you dont access immediatly to BCA protein assay kit, my recomment is to dillute samples, So that prepar your standard on your lysing buffer and add 1 ul to 1 ml of bradford reagent and after 5 min read your sample absorbsion on 595nm, then repeat this method for your samples.this method nearly gives estimated of protein in your samples. Be careful that resete to ziro your double beam spectrophotometer with
unaccompanied RIPA buffer.
I hope this method helpful for you.
Good luck.
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