How is the concentration of purified GFP commonly assessed?

The concentration of purified GFP is often assessed through UV-Vis spectroscopy, particularly at a wavelength around 488 nm. This is because GFP absorbs light in the ultraviolet and visible spectrum, exhibiting a peak absorbance at this wavelength. The amount of light absorbed by the GFP is directly proportional to its concentration, following Beer's Law. This makes it a reliable and straightforward method for quantifying GFP in solution.

The reason other methods might not be the best choice for quantifying GFP concentration includes aspects like specificity and practicality. While measuring fluorescence intensity is indeed utilized, it is more dependent on the excitation and emission wavelengths, which can vary with environmental conditions and may not provide a direct concentration measure without calibration. Mass spectrometry, while accurate for molecular weight determination and identifying protein post-translational modifications, is generally not used for routine concentration assessments due to its complexity and cost. Colorimetric assays could also be used to estimate protein concentration, but they typically require a specific reaction with a dye or reagent that may not be applicable for GFP, owing to its intrinsic fluorescence and specific absorbance characteristics. Thus, UV-Vis spectroscopy at 488 nm stands out as the most common and effective method for this purpose.