After covalently labeling a protein with a fluorescent dye, it is strongly recommended to determine the protein concentration and the labeling efficiency.

What is the degree-of-labeling (DOL)?
The DOL describes how many dye molecules are bound to your protein, e.g. a DOL of 1 refers to a dye:protein ratio of 1:1.
If the DOL is between 0.5 and 1, you should record this number and continue on with your SpS/TRIC measurement.

How do you measure DOL?

To calculate the DOL, use a UV/VIS spectrophotometer to measure an absorbance spectrum of your sample. From this spectrum, read the following values:

• The protein absorbance at 280 nm (A280).
• The wavelength of maximum absorption (λmax). This is a characteristic of the dye used but may vary slightly depending on the buffer conditions and therefore has to be determined experimentally. At λmax, read the absorbance (Amax).

If you’re using a NanoTemper Protein Labeling Kit this calculator can help you calculate the degree-of-labeling (DOL) of a fluorescently labeled protein from absorbance values measured with a spectrophotometer. 

What should the DOL be?
Ideally, it should be between 0.5 and 1. We recommend you record your DOL every time you label your protein and aim to obtain a similar DOL to achieve consistent SpS/TRIC results.

A DOL greater than 1 should be avoided for most proteins since it can lead to adverse effects on protein function. It means that some of your proteins have at least 2 dyes attached (over-labeling) and you’ll need to reduce the ratio of dye to protein. 

A DOL below 0.5 should be avoided since it can lead to a reduced signal-to-noise ratio. It means that your protein is most likely under-labeled and you’ll need to increase the ratio of dye to protein.