Actually I am immobilizing my Enzyme ( an alcohol dehydrogenase ) on Eupergit C 250 L . In order to determine the amount of enzyme that didn't bind to my matrix , I use Bradford assay to determine the protein concentration in my filtrate which become very very diluted especially after washing the matrix.
I think that Bradford assay at these low concentrations is not that much accurate . Any suggestions or recommendations
Hi there,
I suggest to monitor protein concentration at 205nm which is very sensitive as you detect peptide bond absorption at this wavelength. For info:
http://www.ruf.rice.edu/~bioslabs/methods/protein/abs205.html
In my opinion you should try to perform Lowry or BCA assay, both detects low concentration of protein. BCA is 10x sensible than Lowry (aprox)
If you need just a method more sensitive to BCA, lowry or Bradford you can try the nanorange (thermo cod. N6666). Of course the accouracy of any methods depends not only from the protein concentration range, but also from the properties of the buffer that are you using. Otherwise what about pre-concentrate a fraction of you sample by ultrafiltration before the quantification?
There are more sensitive fluorescence-based protein assays than Bradford, Lowry or BCA.See here for an example:
https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-assays-analysis/protein-assays/fluorescent-protein-assays.html
Aside from that, you can improve the sensitivity of the colorimetric assays by concentrating the sample. Manuele Martinelli suggested ultrafiltration. Another way is protein precipitation using the sodium deoxycholate/TCA method. You can, for example, precipitate all the protein from 1 ml of sample in an eppendorf tube, then use the BCA or Lowry assay to measure the amount of protein in the precipitate (perhaps not the Bradford because of the detergent residue). Since these assays can easily detect 2 µg of protein, you can assay as little as 2 µg/ml.
In addition to the above, you can think about concentrating in vacuo, such as on a speedy vac. This can give better recoveries than a precip method. Don't reduce it so far that things start precipitating though. Did you mean 250 ml or 250 l? You can improve the linearity (though not too much affect on sensitivity) of coomassie by using a difference spectrum of bound and unbound coomassie, rather than just 595 nm. This might at least give you a (slightly) better LoQ.
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