MST — Experimental Protocol & Quantitative Analysis

What buffer is used for preparing samples in the MST experiment, and what detergent is added?

GF buffer containing Tween 20 detergent added to a final concentration of 0.05% v/v

What stock concentrations and volumes of Nanobody and EGFP are prepared initially in the protocol?

30 microliters of 5 micromolar Nanobody and 200 microliters of 20 nanomolar EGFP

How is the 16-point serial dilution set up in the PCR microtubes before adding the fluorescent target?

10 microliters of MST buffer is placed in tubes 2 through 16, 20 microliters of 5 micromolar Nanobody is placed in tube 1, and 10 microliters is sequentially transferred and mixed through tube 16

Why must the pipette tip be kept below the meniscus and changed after each transfer step during serial dilution?

Keeping the tip below the meniscus prevents bubble formation, while changing tips after each step prevents carryover of excess protein that would corrupt the two-fold dilution ratio

What volume is removed and discarded from tube 16 after completing the serial dilution?

10 microliters is removed and discarded to waste, leaving exactly 10 microliters of diluted Nanobody in all 16 tubes

How is the fluorescent EGFP target added across the 16 titration tubes?

10 microliters of 20 nanomolar EGFP is added sequentially from tube 16 down to tube 1, mixing gently, which allows the use of a single pipette tip without contaminating lower concentrations

What are the final concentrations of EGFP and the maximum Nanobody concentration in the reaction tubes?

Final EGFP concentration is 10 nanomolar in every tube, and the maximum Nanobody concentration in tube 1 is 2.5 micromolar after 1 to 1 dilution with EGFP

What centrifugation and incubation steps are performed before loading MST capillaries?

Tubes are centrifuged for 2 minutes at top speed in a benchtop microtube centrifuge and incubated for 10 minutes at 25 °C in a heating block

What specific MST instrument parameters and optical settings are used for the Nanobody-EGFP measurement?

Measurement is performed at 25 °C using medium IR laser power, 50% LED excitation power, and blue-channel optics with 470/20 nanometer excitation and 535/60 nanometer emission filters

How is normalized fluorescence (Fnorm in per mille) calculated during MST data analysis in MO.Affinity Analysis?

Fnorm is calculated as the ratio of fluorescence at 5 seconds (hot) divided by fluorescence at 1 second (cold), multiplied by 1000

What is the purpose of switching to delta Fnorm mode in MO.Affinity Analysis software?

It subtracts the Fnorm value of the lowest titrant concentration from every other point, showing the net change in normalized fluorescence across the titration

What regression model and algorithm are used in GraphPad Prism to fit the MST binding curve?

Non-linear regression using the Levenberg-Marquardt algorithm to minimize the residual sum of squares between data points and the 1 to 1 binding model curve

In the 1 to 1 binding equation for MST, which parameters are fixed and which are allowed to vary during fitting?

Total EGFP concentration is fixed to its known value, while Kd, Ffree, Fbound, and the nanobody concentration correction factor n are variable fitting parameters

How are initial estimates for Ffree, Fbound, and Kd obtained before non-linear regression?

Ffree is estimated from the signal at lowest nanobody concentration, Fbound from the highest nanobody concentration, and Kd from the midpoint concentration of the logarithmic sigmoid curve

What do asymmetric 95% confidence intervals from GraphPad Prism indicate about fitted binding parameters?

They indicate how well each parameter is defined by the fit, where narrow intervals mean high precision and wide or undefined limits mean poor parameter definition

When target concentration is below Kd, what percentage of protein is bound at ligand concentrations equal to Kd, 10 times Kd, and 100 times Kd?

50% is bound at 1 times Kd, 91% is bound at 10 times Kd, and 99% is bound at 100 times Kd

What percentage of protein is bound when ligand concentration is 0.5 times Kd, 2 times Kd, and 5 times Kd?

33% is bound at 0.5 times Kd, 67% is bound at 2 times Kd, and 83% is bound at 5 times Kd

According to lab audio notes, why is fluorescence tagging advantageous when measuring binding interactions in complex biological samples?

Fluorescent tags provide high detection selectivity, allowing specific binding interactions to be observed without interference from heterogeneous background molecule