We measure the activity of beta-lactamase enzyme inside bacterial outer membrane vesicles obtained by ultrafiltration of culture supernatants. Beta-lactamase activity value is expressed as mU/mg of protein. For this reason we should measure the protein concentration.
We tried Bradford assay but we obtained unreliable results due to very low protein concentration in our samples. Blank was done with PBS.
We tried also the quantification by Nanodrop but without any significant results.
What other method we might use?
Thank you!
Fluorescence based assays are more sensitive than absorbance-based assays.
https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-assays-analysis/protein-assays/fluorescent-protein-assays.html
The sensitivity of the Bradford or BCA assay can be somewhat increased by increasing the sample volume relative to the dye reagent. This reduces the range of concentrations that can be measured, so the standard curve should be revised to a lower range of concentrations.
I would do what Adam suggested by increasing the sample volume relative to the dye reagent. Another alternative is to quantify by running a WB with a standard curve of your purified and quantified protein and your unknown concentration sample. Comparing the signal from your test sample to the standard curve would give you an estimation of your protein concentration.
HPLC could be a concentrative method. Or µBCA.
For diluted samples another rapid method is to add >50% methanol to sample in a glass tube , centrifugate it after 1H precipitation, carefully discard the supernatant and dry well the tube. A pellet can be observed, but for hydrophobic proteins, a glassy coat could be simply sticked to the bottom of the tube. So, you can directly add the Bicinchoninic acid assay solution to the tube and reveal at 35°C until stable OD signal. With correct standards in parallel of course.
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