I would like to measure protease/amylase/lipase/cellulase activity in culture supernatant of different bacteria. To compare any of these enzyme activity in the supernatant of the different bacteria, I think I have to normalize by protein amount present in the supernatant. Can I use NanoDrop's A280 for the determination of protein in the supernatant?
Nanodrop absorbance is accurate only in the range 0.1-1 as per Lambert-Beer law, so if you have very high absorbance after subtracting the blank, you should consider diluting your supernatant. The blank has to be the same broth you are using to grow the cells.
You can if its just for the purpose of having a normalised number.
but before that, you might wanna try filtering the protein extract/supernatant through a 1.0 um and .4 um before nanodroping it and use the same extraction minus all the protein as blank.
I would also try to dilute and maybe filter before nanodroping. Also, the blank is very important. If you have a culture with the same bacteria strain (so that their metabolism is comparable) but no effect on your protein of interest, then you could use this as blank. Then, the difference will be your protein loss/gain. However, this is a bit tricky, but definitely worth a try.
Yes, I agree with Sherezade Moñino Romero, the 'blank' solution is very important. What blank solution can you use?
Don't you want to compare the enzyme 'activity'? In that case, don't you need to add some kind 'substrate' for these enzyme? How can you measure enzyme 'activity' by measuring A280 (enzyme/protein amount?)?
I just checked online, it looks like 'purified proteins' are a better source for NanoDrop A280 Protein Quantification, NOT the proteins in complex mixtures such as cell extracts or lysates, or probably suspension culutre. According to this website, those samples are best measured using a protein colorimetric assay such as Bradford, BCA, Lowry.
See "NanoDrop Protein Quantification" from ThermoFisher Scintific. [https://www.thermofisher.com/us/en/home/industrial/spectroscopy-elemental-isotope-analysis/molecular-spectroscopy/ultraviolet-visible-visible-spectrophotometry-uv-vis-vis/uv-vis-vis-instruments/nanodrop-microvolume-spectrophotometers/nanodrop-protein-quantification.html]
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