Laboratory Practicals

                  S2005 Methods for characterization of biomolecular interactions

                                       Autumn semester 2023

                  Brno, Masaryk University, Faculty of Science, UKB C4/2^nd floor


During the exercise, we will be analyzing interaction between α-chymotrypsin (CHT, 29.3 kDa) and
soybean trypsin inhibitor (STI, 20.1 kDa). Both stock solutions are prepared in PBS (20 mM
Na-phosphate, 200 mM NaCl, pH 6.7).



MICROSCALE THERMOPHORESIS:

1)     Initial check:

Prior to the affinity measurement, we need to choose the capillaries and check the suitability of
the MST for the CHT-STI system. CHT was labeled in advance using NHS-Red labeling dye (Nanotemper)
according to the product manual.

Sample preparation:

Prepare 500 ul of 10 nM labeled CHT working solution by diluting the stock solution in PBS. Use a
black tube or protect the transparent tube from excessive light exposure.

Prepare 100 ul of 200 μM STI working solution by diluting the stock solution in PBS.

Measurement set-up:

Fill two standard capillaries and, using MO.control SW, perform a preliminary test to check the
fluorescence signal and sticking of protein to the capillaries. If both parameters fulfill the
criteria, continue with the next point. Otherwise, change the conditions (sample concentration,
capillary type) and repeat the experiment.

Perform the binding test. In one tube, mix 30 μl of 10 nM CHT with 30 μl of PBS, and in the second
tube mix 30 μl of 10 nM CHT with 30 μl of 200 μM STI solution. Fill four capillaries with each
mixture and run the experiment.

Data analysis:

Using the MO.control SW, check the obtained data and automated evaluation. Discuss the results.


2)     Affinity measurement:

Once the system's suitability is verified, we can measure the binding affinity.

Sample preparation:

Use the remaining working solutions from preliminary MST tests.

Prepare 16 tubes (use strips) of two-fold STI dilution with the highest concentration of 200 μM and
10 μl final volume in each tube. Add 10 μl of labeled CHT working solution (10 nM) into each tube.

Measurement set-up:

Fill in the information in the MO.control SW. Fill 16 capillaries with the prepared solutions,
place them in the tray in the correct order and start the measurement.

Data analysis:

Using the MO.analysis SW, evaluate the experimental data. Try to calculate the affinity parameters
using default and manual settings. If you have measured duplicate or triplicate, compare the
results and perform a global analysis.


BIO-LAYER INTERFEROMETRY:

1)     Protein immobilization:

The first step in BLI experiment is the immobilization of one binding partner. We will immobilize
STI on AR2G biosensor using amine coupling approach.

Sample preparation:

Dilute the STI stock solution in 10 mM acetate, pH 4.0 to a working concentration of 50 μg/ml and a
final volume 200 μl.

Measurement set-up:

·      Take two unused AR2G biosensors and place them into desired positions of the sensor tray.
Pipet 200 μl of MilliQ water into corresponding positions of hydration plate and insert the plate
into tray.

·      Prepare the experimental plate set-up using Data acquisition SW. Use following steps:

Step

          Solution

                                                      Contact time [sec]

Baseline

          PBS, pH 6.7

                                                      60

Activation

          NHS/EDC mixture (mix immediately before run)

                                                      300

Binding

          STI solution (active sensor)

          Acetate buffer pH 4.0 (blank)

                                                      600

Quenching

          Ethanolamine pH 8

                                                      300

Wash

          PBS, pH 6.7

                                                      120

·      Pipet all solutions into corresponding wells (200 μl per well).

·      Place sensor tray and experimental plate into BLI instrument and run the experiment at 20
°C.

Data analysis:

·      Check the output sensorgrams. Compare the final response of active and blank channel.


2)     Binding affinity assay:

The affinity towards CHT will be determined using direct-binding assay. The dissociation should be
relatively fast and almost complete, therefore, no regeneration step will be introduced in between
measurement cycles.

Sample preparation:

In 1.5 ml tubes, prepare six concentrations of two-fold dilution row of CHT into final volume of
400 μl per tube. The highest concentration shall be 150 μg/ml (app. 5 μM).

Measurement set-up:

·      Take the AR2G biosensors prepared in the previous run and place them into new positions of
the sensor tray. Pipet 200 μl of PBS buffer into corresponding positions of hydration plate and
insert the plate into tray.

·      Prepare the experimental plate set-up using Data acquisition SW. Use following steps for
each of the CHT concentration:

Step

            Solution

                                                       Contact time [sec]

Baseline

            PBS, pH 6.7

                                                       60

Association

            CHT solution of given concentration

                                                       180

Dissociation

            PBS, pH 6.7 (same well as in Baseline step)

                                                       300

·      Pipet all solutions into corresponding wells (200 μl per well).

·      Place sensor tray and experimental plate into BLI instrument and run the experiment at 20
°C.


Data analysis:

·      Using Data analysis SW, check the output sensorgrams.

·      Perform blank sensorgram subtraction.

·      Evaluate the curves using kinetic approach.

·      Evaluate the curves using steady-state approach.

·      Compare and discuss the results.