Introduction for all Labeling Strategies

Detergents are often used to solubilize membrane proteins prior to biochemical investigation. To measure binding affinities using temperature-related intensity change (TRIC) or Spectral Shift, the first step is to label one of the binding partners (the labeled biomolecule is referred to as the target). If the target is a membrane protein solubilized in detergent at a concentration above the CMC, it may be necessary to adapt the labeling protocol. Free detergent micelles can capture excess free dye molecules resulting in 1) a lower dye concentration available in the labeling reaction and 2) the presence of micelle-captured free dye during the binding affinity measurement. This can lead to decreased labeling efficiency and signal quality.

Covalent Labeling Strategies

NanoTemper Technologies covalent labeling protocols (NanoTemper Labeling Kit Protocols) include a separation of free dye from the labeled protein using the B-column. This step is usually sufficient to remove excess free dye, however, if the detergent concentration is above the CMC, free dye captured by micelles will co-elute with the labeled protein and will not be removed (Figure 1).

Figure 1: Schematic representation of standard protein labeling followed by removal of excess free dye using the B-Column (left panel). Membrane protein labeling where the target protein is solubilized in a concentration of detergent greater than the CMC, resulting in excess dye becoming captured in free micelles and co-eluting with the membrane protein following removal of free dye with B-Column (right panel).

Labeling Optimization

There are several suggestions to optimize labeling membrane proteins including detergent considerations, removing free dye by using size exclusion chromatography (SEC) or an affinity tag, and evaluating if the micelles capture dye.   Each suggestion is elaborated on below in more detail if pertinent.

1. Avoid high detergent concentrations during the labeling reaction

2. Perform a Size Exclusion Chromatography step after the labeling reaction

Including a SEC step after incubation of protein and dye allows separation of the labeled protein from dye captured in free micelles as well as the dye alone. Monitoring at 280 nm and 650 nm allows for clear identification of the SEC fractions containing these different species (Figure 2). Further, monitoring 280 nm alone will allow for collection of fractions containing the labeled membrane protein (Protocol: Size exclusion chromatography of membrane proteins to remove free dye captured by micelles protocol).

Figure 2: Size Exclusion Chromatography (SEC) elution profile of a detergent-solubilized membrane protein after labelling using the Protein Labeling Kit RED-NHS 2nd Generation.  Molecules are separated by size and therefore the larger detergent-solubilized membrane proteins elutes earlier than the free micelles. The corresponding peaks are labeled with a representative picture of the respective species.

3. Use a protein tag to remove free dye captured by micelles

If the target membrane protein has a tag that is not used for labeling, it can be used to bind the protein to the corresponding affinity resin after labeling. The micelles that capture free dye during the labeling reaction can then be removed by a washing step before the labeled protein is eluted off the resin.

Figure 3:  Graphic representation of how an affinity tag can be used to remove free dye captured by micelles after a labelling reaction. 1) After labeling, free dye exists in the solution and is captured in free micelles. 2) The available affinity tag on the target protein can be used to bind the labeled target protein to an affinity resin. 3) Washing removes both the free dye and micelle-captured dye. 4) After elution off of the affinity resin, only free micelles and labeled target molecules remain.  

4. Titrate dye with detergent of choice to check if the dye is captured by micelles

NanoTemper RED dyes are sensitive to changes in their chemical environment, therefore, a change in spectral shift ratio (670 nm/650 nm) often occurs if the dye changes from a free state to a micelle-bound state. Titrating the detergent while keeping the dye concentration constant therefore allows determination of the detergent concentration at which the dye binds to micelles. Often, this value will correlate with the CMC (Critical Micelle Concentration (CMC) of LMNG protocol – NanoTemper Technologies).