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Ligand serial dilution

A stepwise dilution of ligand in assay buffer: Typically, a serial 1:1 dilution is performed by transferring one volume of ligand solution to an equal volume of buffer, mixing, and repeating this step. This way, the ligand concentration is reduced by 50% in each dilution step.

See below for a step-by-step illustration of preparing a serial dilution. See further below for preparing a serial dilution of chemical compounds solved in DMSO (or similar), or if your ligand exhibits low solubility in your assay buffer.

 

Serial Dilution of a Ligand in Buffer

General considerations

  • Never prepare less than 20 μl of sample! Otherwise you increase the probability to encounter problems due to evaporation, adsorption of sample material to the plastic micro reaction tubes and higher pipetting errors.
  • Never prepare small volumes (e.g. 20 μl) in large micro reaction tubes (e.g. 500 μl or more)! The high surface to volume ratio leads to surface adsorption even for well-behaved proteins. Always use the smallest micro reaction tubes possible (e.g. PCR tubes).
  • Always mix such small volumes with a pipette. Do not vortex! Do not flip the tube!
  • Avoid any buffer dilution effects! The buffer in tube 1 and the buffer in the tubes 2-16 must be the same. Otherwise you get a gradient in salt, DMSO, glycerol or other additives. This will bias the MST measurements.

1. Prepare a serial dilution of the ligand using the ligand dilution buffer

  • Transfer 20 µl of ligand into tube 1.
  • Transfer 10 µl ligand dilution buffer into tubes 2 to 16.

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  • Now transfer 10 µl of ligand from tube 1 to tube 2 and mix carefully by pipetting up and down.
  • Transfer 10 µl from tube 2 to tube 3.
  • Continue the serial dilution up to the final step where you transfer 10 µl from tube 15 to tube 16.
  • Discard 10 µl from tube 16 to get an equal volume of 10 µl for all samples.

2. Add 10 µl of your target to each tube from 16 to 1 and mix by pipetting

 

3. Dip a Monolith NT.115 capillary into each tube from 1 to 16, put them in positions 1 to 16 of the device tray and start the measurement

 

Serial Compound Dilution in DMSO (or other organic solvents)

Compounds that are dissolved in DMSO can be diluted as described under serial dilution of a ligand in buffer (making sure of course that the final DMSO concentration is constant throughout the dilution series). However, dilution of organic compounds into buffer can sometimes result in compound precipitation. A serial dilution of a precipitated compound will have a large error. Instead, one can first prepare a dilution series of the compound in 100% DMSO and then dilute all samples into assay buffer in a second dilution step. This ensures ligand solubility and protects the target from high DMSO concentrations. Please note that the instructions below will differ from those on the instructions page in the MO.Control software.

Note: If following this protocol, which ensures a DMSO percentage of 2% (as opposed to 5%), do not select "ligand in organic solvent like DMSO" on the plan page and disregard the instructions for ligand dilution given in MO.Control. 

1. Prepare a serial dilution of the compound using 100% DMSO

  • Transfer 10 µl of compound (dissolved in 100% DMSO) into tube 1.
  • Transfer 5 µl DMSO (100%) into tubes 2 to 16.
  • Now transfer 5 µl of compound from tube 1 to tube 2 and mix carefully by pipetting up and down.
  • Transfer 5 µl from tube 2 to tube 3.
  • Continue the serial dilution up to the final step where you transfer 5 µl from tube 15 to tube 16.
  • Discard 5 µl from tube 16 to get an equal volume of 5 µl for all samples.

2. 2nd dilution of compound in buffer

Prepare 16 tubes with 48 µl assay buffer and transfer 2 µl from the tubes from step 1 to the corresponding buffer tube (e.g. transfer 2 µl of DMSO-dissolved ligand from tube 1 from step 1 into tube 1 from step 2 which contains 48 µl assay buffer. The resulting DMSO concentration will be 4%).

 

3. Prepare target molecule

Prepare 16 tubes containing 10 µl of labeled target molecule each.

 

4. Mix 10 µl of your diluted compound with 10µl of target

Transfer 10 µl of the serial compound dilution from step 2 to the 16 tubes containing your target from step 3. Note that the final DMSO concentration has decreased to 2% in the assay. The highest ligand concentration is 50-fold more dilute than the stock concentration (25-fold dilution in step 2 and 2-fold dilution in step 4).

 

5. Dip a Monolith capillary into each tube from 1 to 16 (step 4), put them in positions 1 to 16 of the device tray and start the measurement

 

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