Magnetic Tweezers in a Microplate Format

Summary

Here, we describe the use of a novel microplate assay to enable mechanical manipulation of biomolecules while performing ensemble biochemical assays. This is achieved using a microplate lid modified with magnets to create multiple static magnetic tweezers across the microplate.

Abstract

Mechanobiology describes how the physical forces and mechanical properties of biological material contribute to physiology and disease. Typically, these approaches are limited single-molecule methods, which restricts their availability. To address this need, a microplate assay was developed that enables mechanical manipulation while performing standard biochemical assays. This is achieved using magnets incorporated into a microplate lid to create multiple magnetic tweezers. In this format, force is exerted across biomolecules connected to paramagnetic beads, equivalent to a typical magnetic tweezer. The study demonstrates the application of this tool with FRET-based assays to monitor protein conformations. However, this approach is widely applicable to different biological systems ranging from measuring enzymatic activity through to the activation of signaling pathways in live cells.

Introduction

Mechanobiology focuses on understanding how the propagation of physical forces within and between cells regulates cellular activity^1,2 and how this correlates with the organization and dynamics of both proteins and cells.

Single-molecule force measurements have revealed how force is used in biological systems, from single proteins to whole cells and tissues^3,4,5,6,7. These challenging experiments require specialized equip....

Protocol

Sample proteins required for this experiment and a list of reagents are found in the Table of Materials. Equivalent proteins should be produced for the user's system of study to measure conformation changes.

1. 3D printed magnetic lid

1. Design a microplate lid to house the magnets within a 24-well microplate. An example CAD file can be downloaded from GitHub¹². The measurements are shown in Figure 1.
   NOTE: The height of the pedestal can be altered to change the distance between magnets and surface to alter the applied force. In addition, t....

Results

Figure 2 shows an example of a well-scan measurement where the fluorescence intensity of GFP has been recorded at 1 mm intervals across the microplate well. Typical fluorescence measurements are performed at the center position of the microplate well (position 8,8 in Figure 2); it is, therefore, important that there is bound protein at this location. As shown in Figure 2, the intensity is highest in the center of the well within a r.......

Discussion

This approach enables force-based measurements to be readily applied in a microplate using fluorescent plate readers. Importantly, this assay format assumes there is functional protein when it is bound to a surface. Therefore, prior knowledge is required before embarking on these measurements to ensure there is protein activity. It is also beneficial to make sure that the binding of molecules to the paramagnetic beads and surface is optimized for each system.

This concept can be modified to f.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
24 well glass-bottom microplate	Cellvis	P24-1.5H-N	Multiple sources are available. Unless needed, it is best to avoid treated surfaces and we use Imaging grade glass N1.5.
Anti-RFP antibody	Abcam	ab290	Multiple sources are available but must ensure there is minimal reactivity with GFP.
Bench top light microscope	Optika	IM-3
Bench top Rotator	Cole-Palmer-Stuart	SB3
Biotin-BSA	Sigma Aldrich	A8549
CAD Software - Sketch Up Educator	Sketch Up		Alternative CAD softwares can be used. Users should ensure the file formats are compatiable with their 3D printer.
Dynabeads Protein A	Fisher Scientific	10746713	2.8 µm paramagnetic beads with recombinant Protein A
Impact contact adhesive	EVO-STIK
MagnaRack magnetic separation rack	ThermoFisher Scientific	CS15000	Magnetic Isolator
NaCl	Fisher Scientific	10316943
Neodymium N42 5mm cube Magnets	Supermagnete	W-05-N
Plate Reader - ClarioStar	BMG Labtech		All plate reader systems can be used where measurements are possible from under the microplate. The magnet lid excludes standard measurements from above
Streptavidin	Sigma Aldrich	189730
Tris-HCl	Fisher Scientific	10142400
Ultimaker PETG Filament	Ultimaker
Ultimaker S3 - 3D printer	Ultimaker