top of page
Search

Microscale Thermophoresis (MST) Protocol

A standard 16-point binding assay using MST


First-time users, please contact Mr Pengjun Xia (pjxia@udel.edu) in the Parashar laboratory.



The following protocol describes the labeling procedure for and binding assay using 16 capillaries in an MST experiment. The volumes can be up- or downscaled as needed



Materials

  • Nuclease free water

  • 5X PBS-T buffer

  • Dye Stock

  • Protein and ligand stocks

  • Centrifuge

  • PCR tubes (or 384-well multi-well plates with a non-binding surface)

  • Capillaries

  • Pipette and tips


Section 1: PROTEIN LABELING

Following protocol for labeling a His-tagged protein using RED-tris-NTA dye

  1. Add 8.0 mL nuclease-free water to the vials containing 5 x PBS-T to obtain 1 x PBS-T.

  2. Suspend the dye in 50 uL of PBS-T to obtain a 5 uM dye solution.

  3. Prepare a 100 nM dye solution by mixing 2 ul of dye (5 uM) and 98 uL PBS-T (or your buffer).

  4. Adjust the protein concentration to 200 nM in a volume of 100 uL.

  5. Mix 100 uL of protein (200 nM) with 100 L of dye (100 nM).

  6. Incubate for 30 minutes at room temperature.

  7. Centrifuge the sample for 10 min at 4 C and 15 000 g.

  8. The protein is labeled and ready for the binding assay.


Section 2: BINDING ASSAY

Prepare a serial dilution in PCR tubes (or in 384-well multi-well plates with a non-binding surface) using the following method:

  1. Prepare 20 uL of the ligand at 2 x concentration (e.g, for a final concentration of 500 nM, prepare ligand working stock at a concentration of 1000 nM) in tube 1 (see picture below).

  2. Add 10 uL of PBS-T (your buffer or water) into the wells/PCR tubes 2-16 (see picture below).


3. Transfer 10 uL of the ligand from well/PCR-tube 1 to well/PCR-tube 2 with a pipette and mix by pipetting up-and-down multiple times (be gentle; avoid frothing).

4. Transfer 10 uL to well PCR-tube 3 and mix. Repeat the procedure for well/PCR-tube 4-16. Discard the extra 10 uL from well/PCR-tube 16.

5. Load the capillaries and measure the samples using MO. Control software and analyze data using MO. Affinity Analysis software.

 

Tips

1. Avoid touching the center of the capillaries.

2. Have 0.05% Tween20 in your buffer.

3. Always optimize your buffer (see buffer compatibility chart below) as well as conc. of proteins and ligands.

4. Start with auto-excitation power and try different MST power settings (i.e., low, medium, and high).

5. Once the optimal excitation power and MST power are determined, measure your samples 2 more times.

 

Buffer Compatibility


 

Useful Videos from YouTube





Comments

bottom of page