Hello,
I'm wondering about the best method to measure the protein concentration in protein samples (Tissue samples and cell culture samples) extracted with Ripa buffer (Sigma-Aldrich, R0278).
We have tried using the NanoDrop (ND-1000) and the Protein Quantification Kit - Rapid from Sigma (based on Coomassie blue, Sigma-Aldrich, 51254). The NanoDrop seemingly can't cope well with the viscose solution, so one and the same sample can have quite good or any concentration, including negative values, if measured several times in sequence. The Sigma kit unfortunately reacts with the Ripa buffer itself, giving us a standard curve with a too shallow slope to calculate correct protein contents.
Does anyone have a good suggestion of which alternative quantification method to use together with Ripa buffer?
Alternatively, I can extract protein from small samples with the Qiagen AllPrep kit (Qiagen, 80284). Which buffer would you suggest to dissolve the protein pellet in afterwards? (However, I need to preserve the sample for future testing as well, e.g. freeze it.)
Thanks a lot!
Hello Nadine
If your samples are viscous post cell lysis in RIPA buffer, its most likely due to the released DNA. This can easily be dealt with by sonication of your samples (I do not recommend DNAse I since you want to estimate protein concentration later). What I observed was that one needs a blank containing RIPA buffer as well, if you plan to use Nanodrop (Absorbance at 280nm). Another thing that worked for me was to dilute the lysate (post sonication) prior to measurement, especially if the lysate has a a lot of protein and the dilution also helps to reduce the detergent concentration with respect to that in 1x RIPA.
Hope this helps
RIPA buffer contain SDS that can interferee with some colorimetric assays.
As reported in the attached file you can try the BCA assay (that you can acquire from Thermo.
https://www.thermofisher.com/it/en/home/life-science/protein-biology/protein-assays-analysis/protein-assays/colorimetric-protein-assays/bca-protein-assays.html?gclid=Cj0KEQjwmIrJBRCRmJ_x7KDo-9oBEiQAuUPKMjLKu_R11NUiEkchiFxIDpRNf82N9mwOGtuOkvXVw10aAnq38P8HAQ&s_kwcid=AL!3652!3!92390238014!b!!g!!%2Bbca%20%2Bassays&ef_id=WNTxyQAAAcR3kSMA:20170522165243:s)
You can certanilly optimize sample preparation for nanodrop measurment as Aparajita suggest to you, but if it is certanilly cheap i think that could be difficult if you are working with small volumes and more time expensive.
good luck
Manuele
I would second Manuele. We have had good experience with this BCA kit and the RIPA buffer. An addition consideration if the readings are still inconsistent is to try and make a dilution curve of something simple like BSA dissolved in the buffer and diluted by TBS or PBS. This should tell you a useful dilution to work with.
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