Molecular Spring Constant Analysis by Biomembrane Force Probe Spectroscopy

A biomembrane force probe (BFP) is an in situ dynamic force spectroscopy (DFS) technique. BFP can be used to measure the spring constant of molecular interactions on living cells. This protocol presents spring constant analysis for molecular bonds detected by BFP.

Transcript

This video describes step-by-step procedures on using Biomembrane Force Probe, or BFP to measure molecular spring constant. In force probe, BFP has recently emerged in the native cell surface or in SITU Dynamic Force Spectroscopy analysis. The main aim of this technique is to measure single molecule binding genetics.

This data analysis procedure is a simple but powerful message and provide mechanical information, specifically the dynamic force and inflammation of the cell membrane expressing molecules. During the BFP experiment cycle, the probe microbiome bet with the red blood cell and probate, the target cell and the ligand receptor molecular bonding between the probe and the target can be considered as a series connected brain system. Based on Hooke's law, the reciprocal of the total spring constant of a series connected spring system is equal to the sum of the inverse spring constant of each single component, as the equation shows.

The spring constant of red blood cell is calculated based on events motion, which is dependent to the radial of the probe micropipette in the orifice. The red blood cell, the circular contact area between the red blood cell and the probate and the micro-pipette aspirating pression. The BFP road data is obtained by a homemade left view virtual instrument, which controls the movement of the target micro-pipette by a PAs Translator.

A full PFP touch cycle consists of five stages.Approach.Impinge.Contact. Retract and dissociate. In the beginning, does stress the Robusto APICS position is denoted as X equals 0 in black deshine.

The target is then driven by a piece or translator to impinge on and compress the robusto to cause negative probate movement. You note it as X smaller than zero in red dash line. In the retract stage, the red busto apex move from the X smaller than zero position back to the X equal to zero position referred to as the compressive phase.

If a bond is formed between the ligand receptor molecule complex, the red blood cell would further different to other positive direction. This X greater than zero period is referred to as the tensile phase of the retract stage. The retract stage is the most critical part to determine the spring constant of the molecule of bonding.

Collect force versus time road data using the BSP data acquisition platform. Each experiments usually record 50 to 200 touch cycles, Open the PFP data analysis software. Click on the yellow folder icon and select the corresponding row data file by double clicking on them.

We're on the program. Then click on the up and down arrow buttons to switch between events. Using the outlier exclusion criteria to screen out invalid events.

Select the exporting data type that's forced to time format and choose the appropriate state range of time. Click on the export plot data button. The exported data is saved as text file in default.

This text file contains two columns of data. It's the first column representing the time daddies and the second column representing the corresponding force at each time point. Plot the force versus time curve using spreadsheets software to obtain the forced versus displacement curve.

Multiply the time value of each data point by the Willow Steel PSO movement. Zero the first data point by subtracting the smallest displacement value from each data point. This horizontal transformation does not affect the subsequent spring constant calculation.

In the force versus displacement curve, two distinct actor group was different if the new force slope can be identified. Each representing the compressive phase and the 10 style phase. Fit a regression line to each data group.

The line with steeper slope represents the total spring constant at a compressor phase denoted as K1.And the line that was smallest top represents the line at this 10 style phase denoted as K2.As I've mentioned, the total spring constant is the reciprocal of the son of the series spring constant of each component. During the compressive phase of the beat to cell mode, the molecular bonding is not stretched, therefore the molecular bonding spring constant is not taken into consideration. The spring constant of the target cell is described as the equation displayed where K1 represents the total spring constant during the compressor phase.

In the 10 style phase of the beat to sell mode, the total spring constant is the sun of the uverse spring constant of the red blood cell molecular bond. And the target cell, use the equation displayed to calculate the moleculer bonding spring constant, which K2 represents the total spring constant. You read the 10 stop phase.

In the beat to beat mode, since B deformation is negligible, the term which describes the inverse of the target cell spring constant approach zero, therefore the total spring constant in the compressor phase is equivalent to the red blood cell spring constant showing as the following equation. In the tensile phase, the spring constant of the molecular bonding can be calculated by subtracting the effect of the red blood cell showing as the following equation. Collect spring constants of the side-wind integrating attitude B, Beta 3 complex, the K562 cell and the Febrifugine integrating attitude B Beta three complex, calculate the mean and standard deviation of the derived spring constants at pick a Newton or nanometers scale.

Now you should have a better understanding of what are the requirements for measuring and spring constant out of the VFD data. The differences between the beat to cell mode and the beat to beat mode should be noticed. Different molecule presenting service will have different defenses to the measure of spring constant.

In conclusion, this step-by-step procedure describes how to perform molecular spring constant analysis using BFP. We foresee future efforts will be made to automate and integrate the BFP data acquisition and DFS analysis into one computer rest program. Making the entire BFP operation data analysis more user friendly and high throughout.

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Journal (Bioengineering)

Journal (Bioengineering)

Molecular Spring Constant Analysis by Biomembrane Force Probe Spectroscopy

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