I want to measure the protein concentration C, and I know that my protein of interest is the only protein that I have in solution. Why don't people use intrinsic Phe, Tyr, Trp fluorescence for this purpose?
The most common ways of protein concentration measurements are:
If the protein has a reasonable number of aromatic residues, it will exhibit high fluorescence signal in the UV range. Then why don't we calibrate this signal to measure C? This approach seems far more sensitive than (1), and it does not require any additional reagents as (2) and (3)
Hi there,
Calibration of the fluorescence signal of a particular protein supposes that you have the pure protein in hand with an already known concentration (determined by UV or Bradford). As fluorescence is intrinsecally protein specific and also depends on the actual state of the protein, it will make things difficult...
In addition to Prof. Liger mentioned, I would like to add that oligomeric status of the protein also affects its intrinsic fluorescence. If your protein may have more than one oligomeric forms in solution, it will be difficult to interpret the results. The absorbance at 280 using molar extinction coefficient is really sensitive to estimate accurately.
Fluorescence is not normally used for quantification because there are many factors that affect it. Proteins must be in a buffered medium that affects quantum yield. On the other hand, each of the different protein residues (aminoacids) has a very different behavior (different excitation and emission), so it is not possible to carry out this type of measurement. Perhaps, for very small peptides and in which the primary structure is perfectly known, it could be tried, but in other cases it is not possible.
Protein fluorescence is in essence caused by Trp, and the intrinsic Trp-fluorescence depends on the hydrophobicity of its micro-environment, as others have noted.
If you want to use fluorescence, why not use established procedures with fluoram, OPA or epicoccone?
UV- Vis is a standard option which is more feasible and accessible. Fluoroscence, on the other hand, is preferred for certain amino acids.
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