This work was supported by the Burroughs Wellcome Career Award at the Scientific Interface (A.R.D.), the National Institutes of Health through the NIH Director's New Innovator Award Program 1-DP2-OD007078 (A.R.D.), the William Bowes Jr. Stanford Graduate Fellowship (A.S.A.), and the Stanford Cardiovascular Institute Younger Predoctoral Fellowship (J.C.). The authors thank James Spudich for loaning microscopy equipment.

1. Flow Cell Preparation
<ol>
	<li>Coverslips (#1.5, 22x22 mm and 22x40 mm, VWR) are cleaned using sonication.
	<ol>
		<li>Add the coverslips to a small glass container capable of holding coverslips and fitting in the sonicator (see step 2).</li>
		<li>Fill the container with isopropanol and sonicate in a bath sonicator for 20 minutes.</li>
		<li>Discard the isopropanol and rinse the coverslips with copious quantities of deionized water produced by a Barnsted MilliQ apparatus or similar device. Fill the container with ...

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No conflicts of interest declared.

This protocol describes a new use for a classical single molecule technique. Magnetic tweezers allow medium to high-throughput single molecule assays in a cost-efficient manner. However, like all experimental techniques there are challenges and potential pitfalls.
Limitations of magnetic tweezers
Compared to an optical trap the spatial and temporal resolution of a MT apparatus is low. Moreover, the forces generated by the simple MT described here are 30 pN or less, sig...

<table border="1">
 <tbody>
 <tr>
 <td>Name of Reagent</td>
 <td>Company</td>
 <td>Catalogue Number</td>
 </tr>
 <tr>
 <td>Micro Cover Glass #1.5 (22x22)</td>
 <td>VWR</td>
 <td>48366-067</td>
 </tr>
 <tr>
 <td>Micro Cover Glass #1.5 (22x40)</td>
 <td>VWR</td>
 <td>48393-048</td>
 </tr>
 <tr>
 <td>Lambda DNA</td>
 <td>Invitrogen</td>
 <td>25250-010</td>
 </tr>
 <tr>
 <td>T4 DNA Ligase</td>
 <td>Invitrogen</td>
 <td>15224-041</td>
 </tr>
 <tr>
 <td>Microcon Ultracel YM-100</td>
 <td>Millipore</td>
 <td>42413</td>
 </tr>
 <tr>
 <td>Anti-Digoxigenin</td>
 <td>Roche Diagnostics</td>
 <td>11-333-089-001</td>
 </tr>
 <tr>
 <td>Tween 20</td>
 <td>Sigma</td>
 <td>P9416-100ML</td>
 </tr>
 <tr>
 <td>Anti-myc Antibody</td>
 <td>Invitrogen</td>
 <td>46-0603</td>
 </tr>
 <tr>
 <td>Bovine Serum Albumin</td>
 <td>Sigma</td>
 <td>B4287-5G</td>
 </tr>
 <tr>
 <td>Dynabeads M-280 Streptavidin</td>
 <td>Invitrogen</td>
 <td>658.01D</td>
 </tr>
 <tr>
 <td>Dynabeads MyOne T1 Streptavidin</td>
 <td>Invitrogen</td>
 <td>658.01D</td>
 </tr>
 <tr>
 <td>p-Aminophenylmercuric Acetate</td>
 <td>Calbiochem</td>
 <td>164610</td>
 </tr>
 <tr>
 <td>Biotin-Maleimide</td>
 <td>Sigma Aldrich</td>
 <td>B1267</td>
 </tr>
 <tr>
 <td>Biotin labeled oligo</td>
 <td>IDT DNA</td>
 <td>Custom synthesis</td>
 </tr>
 <tr>
 <td>Digoxigenin labeled oligo</td>
 <td>IDT DNA</td>
 <td>Custom synthesis</td>
 </tr>
 <tr>
 <td>Collagen peptide gene</td>
 <td>DNA 2.0</td>
 <td>Custom synthesis</td>
 </tr>
 <tr>
 <td>MMP-1 cDNA</td>
 <td>Harvard Plasmid Database</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>z-translator</td>
 <td>Thorlabs</td>
 <td>MTS50</td>
 </tr>
 <tr>
 <td>Servo controller for translator</td>
 <td>Thorlabs</td>
 <td>TDC001</td>
 </tr>
 </tbody>
 </table>

multiplexed single-molecule force proteolysis measurements using magnetic tweezers

The generation and detection of mechanical forces is a ubiquitous aspect of cell physiology, with direct relevance to cancer metastasis1, atherogenesis2 and wound healing3. In each of these examples, cells both exert force on their surroundings and simultaneously enzymatically remodel the extracellular matrix (ECM). The effect of forces on ECM has thus become an area of considerable interest due to its likely biological and medical importance4-7.
	Single molecule techniques such as optical trapping8, atomic force microscopy9, and magnetic tweezers10,11 allow researchers to probe the function of enzymes at a molecular level by exerting forces on individual proteins. Of these techniques, magnetic tweezers (MT) are notable for their low cost and high throughput. MT exert forces in the range of ~1-100 pN and can provide millisecond temporal resolution, qualities that are well matched to the study of enzyme mechanism at the single-molecule level12. Here we report a highly parallelizable MT assay to study the effect of force on the proteolysis of single protein molecules. We present the specific example of the proteolysis of a trimeric collagen peptide by matrix metalloproteinase 1 (MMP-1); however, this assay can be easily adapted to study other substrates and proteases.

Watch this Scientific Journal Video about Multiplexed Single-molecule Force Proteolysis Measurements Using Magnetic Tweezers at JoVE.com

Multiplexed Single-molecule Force Proteolysis Measurements Using Magnetic Tweezers

In this article we describe the use of magnetic tweezers to study the effect of force on enzymatic proteolysis at the single molecule level in a highly parallelizable manner.

The  generation and detection of mechanical forces is a ubiquitous aspect of cell physiology,  with direct relevance to cancer metastasis1, atherogenesis2 ...

multiplexed-single-molecule-force-proteolysis-measurements-using

Research

JoVE Journal

Bioengineering

11.5K vistas.  Stanford University. En este artículo se describe el uso de pinzas magnéticas para estudiar el efecto de la fuerza en la proteólisis enzimática a nivel sola molécula de una manera altamente paralelizable.