How to Measure the Extinction Coefficient of Fluorescent Proteins
The determination of the extinction coefficient of fluorescent proteins is conducted using a spectrophotometer, which evaluates their concentration based on the absorbance of the fluorescent protein at a specific wavelength. The general steps of the determination process are as follows: calculation of the theoretical extinction coefficient, sample preparation, and application of the Beer-Lambert law.
1. Calculation of Theoretical Extinction Coefficient
First, calculate the theoretical extinction coefficient based on the amino acid sequence of the fluorescent protein (FP).
2. Sample Preparation
Next, prepare the purified FP sample. Use a spectrophotometer to measure the absorbance of FP at its absorption peak.
3. Application of the Beer-Lambert Law
Finally, apply the Beer-Lambert law to calculate the extinction coefficient using the formula: A = εlc, where A is the absorbance, ε is the extinction coefficient, l is the path length, and c is the sample concentration. If the values of A, l, and c are known, ε (extinction coefficient) can be calculated.
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