I am wondering if anyone on here has experience using native fluorescence/HPLC to quantify protein concentration. Up until now we've been using ELISAs as the protein quantities are in the ng/pg range, but obviously HPLC would make things much easier/quicker. The proteins we are assaying are about 30 kDa., and have the expected number of aromatic residues.
We have an Aglient 1100, and I'm looking at purchasing a used fluorimeter to eliminate the need for doing ELISAs; however, given that we can't label the proteins (I'm assuming variations in labeling efficiency between the calibration standards and the assayed samples would be too great) I'm concerned whether native fluorescence would be sufficiently sensitive. Is this likely to be the case?
On a related note, is there a web server that can analyze amino acid sequence and predict improvement in signal when comparing native fluorescence to absorbance at 220nm?
Any feedback would be greatly appreciated.