INTRODUCTION:
Knowing the amount of dye conjugated to a protein is essential for predicting the amount of dye required for an experiment and for ensuring good control of fluorescence between experiments.
The following blog post explains how to calculate the degree of protein labeling by a selection of dyes by using protein and dye absorbances.
Important
- All unbound dye must be removed before determining absorbances. This should be done by extensive dialysis or gel filtration
- The extinction molar coefficient (ε) of the unlabeled protein is required
- The extinction molar coefficient and the wavelength the fluorescent dye absorbs maximally at is also required. The table below shows some routinely used dyes.
- A correction factor is also required as protein concentration is determined at 280nm, however the dyes also absorb at 280nm. The correction factor is the A280 of the dye divided by the Amax of the dye.
Critical Values for commonly used dyes:
| Fluorescent Dye | Wavelength Max. (λmax) (nm) |
Extinction Coefficient (ε') (M-1cm-1) |
Correction Factor |
| FITC | 494 | 68,000 | 0.300 |
| TRITC | 555 | 65,000 | 0.340 |
| NHS-Rhodamine | 570 | 60,000 | 0.340 |
| Texas Red Sulfonyl Chloride | 595 | 80,000 | 0.180 |
| R-Phycoerythrin | 566 | 1,863,000 | 0.170 |
Calculate Degree of Labeling
Remove all excess, unbound dye by dialysis or gel filtration.
Measure absorbance at 280nm and the wavelength max (Amax) in a cuvette with a 1cm pathlength.
NOTE: If absorbance is >2 then dilute the solution to achieve values <2.0.
Calculate molarity of the protein:
- ε = protein molar extinction coefficient
- Amax = Absorbance of dye measure at wavelength max.
- CF = Correction factor
- Dilution factor = Amount, if any, sample was diluted before absorbance readings were taken
Protein Concentration (M) = ((A280 -(Amax x CF)) ÷ ε) X Dilution factor
Calculate the degree of labeling:
- ε' = dye molar extinction coefficient
