Attachment of Anti-GFP Antibodies to Microspheres for Optical Trapping Experiments

Abstract

In vitro motility assays enabled the analysis of coupling between ATP hydrolysis and movement of myosin along actin filaments or kinesin along microtubules. Single-molecule assays using laser trapping have been used to obtain more detailed information about kinesins, myosins, and processive DNA enzymes. The combination of in vitro motility assays with laser-trap measurements has revealed detailed dynamic structural changes associated with the ATPase cycle. This protocol describes a method for attaching anti-GFP (green fluorescent protein) antibodies to microspheres. GFP-motor fusion proteins can then be adsorbed to the microspheres for use in single-molecule motility studies and optical trapping experiments.

MATERIALS

It is essential that you consult the appropriate Material Safety Data Sheets and your institution’s Environmental Health and Safety Office for proper handling of equipment and hazardous materials used in this protocol.

Reagents

• Anti-GFP antibody (mouse monoclonal, 0.05 mg/mL in PBS [mAb3E6]; Qbiogene/MP Biomedicals or similar)

• Beads (carboxylated microspheres, 0.5- to 1-μm diameter; Polysciences or similar)

• Bovine serum albumin labeled with tetramethyl rhodamine isothiocyanate (TMR-BSA)

• Phosphate-buffered saline (PBS; 100 mM)

Equipment

• Centrifuge

• Incubator preset to 22°C

METHOD

1. Dilute 10 μL of beads in 100 μL of PBS. Centrifuge at 20,000g for 1 min at 22°C.

2. Resuspend beads in another 100 μL of PBS and repeat Step 1.

3. Resuspend beads in 20 μL of anti-GFP antibody +1 μL of TMR-BSA. Incubate for 5 min at 22°C.

4. Centrifuge beads at 20,000g for 1 min at 22°C.

5. Resuspend beads in 20 μL of PBS.

6. Repeat Steps 4 and 5 three times.