FAQ

How does the Fluidity One compare to A280 on a NanoDrop or UV spectrophotometer?

The absorbance at 280 nm is directly proportional to protein concentration, and is produced by tryptophan, tyrosine and cystine residues (in order of decreasing impact). Ideally the measurement is made on denatured protein (in 6 M guanidine hydrochloride), although in practice measurements are typically made on the protein in its native state. This was shown by Gill and von Hippel (Anal. Biochem. (1989), 319-326) to result in an average standard deviation of ± 3.8% with a maximum deviation of + 14.9% for a set of 19 different proteins. The error in the spectrophotometric measurement itself is typically 1-3%, although this is highly dependent on the instrument and the sample concentration. A280 measurements are only useful down to a protein concentration of approximately 150 nM (depending on the protein).

The Fluidity One, on the other hand, carries out a labelling reaction to render the examined protein(s) fluorescent. The latent fluorophore reacts with amine groups (ie lysine residues and chain N-termini), thus the fluorescent yield of a protein is proportional to the number of lysines and chains in the protein.  Using our fluorescent labelling system we are able to detect proteins down to a concentration of about 10 nM (depending on the protein), so we offer 15-fold greater sensitivity than A280 measurements. 

For further discussion and comparison of the two techniques, see our blog post here.

You can also see our study comparing the Fluidity One to the A205 method here.

Back to FAQs