If you’re using a NanoTemper Monolith 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.
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 MST measurement.
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 MST 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.
Well to calculate DOL, we needed to calculate the protein concentration too. So instead of just keeping your protein’s concentration to ourselves, we thought you might like to know it as well!