Do you have potassium in your sample/buffers? Also could be a salt formed between copper sulfate from the protein assay and SDS (or possibly the deoxycholate) from RIPA. If you don't need the nuclear proteins, DNA or the lipid rafts from the cell membrane, try using Tween 20 or TX100 instead as detergent.

Dear all, please have a look at the following similar RG thread. My Regards

https://www.researchgate.net/post/Why_did_I_get_a_precipitate_of_my_protein_samples_when_added_to_bradford

Hello Samina Momtaz

The detergents present in RIPA buffer interferes with the protein assay. RIPA buffer contains a mixture of ionic and non-ionic detergents. A classical RIPA buffer formulation contains 1% w/v of NP40/Igepal (non-ionic detergent) and a mixture of 0.5% sodium deoxycholate and 0.1% SDS (ionic detergents). Moreover, RIPA buffer has a low solubilizing power.

You could precipitate the proteins in the sample so as to remove the interfering substances, and then re-dissolve the protein pellet in an assay-compatible buffer and perform the assay.

Best.

Hi Malcolm Nobre,

Thank you for your response. We also think that it might be due to the detergent in the RIPA. We're using the RIPA formulation as you mentioned. Are you suggesting precipitating the protein lysate in the sample buffer? Should I use the RIPA for lysis or any other buffer?

Thanks,

Samina.

Hi Samina

It has been suggested that oligosaccharides such as cyclodextrin can be used to overcome difficulties associated with detergents and surfactants interfering with the color changes of protein-staining dyes.

It is believed that the detergent forms a complex with the cyclic oligomer. By using a suitable amount of one or more cyclic oligomers in the detection reagent, the detergent can be trapped in an oligomer-detergent complex, thereby allowing its separation from the protein.

Please refer to the link below for more information.

https://patents.google.com/patent/US9091694B2/en

You may also obtain some more information on this subject from the paper attached below.

https://www.sciencedirect.com/science/article/abs/pii/S0003269776800644

Hope this helps!

Best.

Hi Malcolm Nobre

Thank you for your explanation. I will follow the articles you linked here.

Best,

Samina.