I prepared RIPA buffer to lysis my cells and went for protein assay, i added my protein samples to bradford right before measurging OD, but i didnt get homogenic mixture. i got a blue precipitate. tried to repeat with different bradford and got the same result. then thought it might be some dna fractions inside my samples, so i centrifuged but samples are clean.
Hi there,
If your sample is too concentrated in proteins you get precipitation. I suggest you to dilute the sample prior to protein assay.
You could also use Pierce 660nm protein assay instead of Bradford, quick assay with high tolerance towards the detergents.
BioRad's own troubleshooting guide for the Bradpord assay states that detergents in protein solutions will cause precipitates (see http://www.bio-rad.com/webroot/web/pdf/lsr/literature/4110065A.pdf).
You can a) dialyse to remove detergent, b) dilute protein in a non detergent buffer (high salt not a good idea) or c) use a detergent compatible test such as the Lowry (also marketed by BioRad as a DC-compatible protein test).
www.malariaresearch.eu
Hi there,
I believe you are facing solubility issue. If the proteins are precipitates due to insolubility. Its generally prescribed to add of 1M NaOH to solubilize the proteins in the wells of protein sample and also blank controls.
I believe this has addressed your query.
Rajkumar
actually this precipitation was due some detergent as some of you have mentioned, dilution of samples does help with precipitation
Thank you all
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