Single bead
Department of Physics, Willamette University
Single Bead Optical Trapping Assay Using GFP-tagged Motor
Before you start, prepare an aliquot of α-GFP/10 (5 μL of α-GFP 0.5 mg/mL stock + 45 μL AB)
Make α-GFP beads
1. Take 10 μL of 1-μm diameter carboxylated beads
2. Wash beads 3x in AB All wash steps should be performed as follows
- Spin down beads in a benchtop centrifuge at max speed (14k rpm) for 1-2 minutes
- Carefully, pull off the supernatant
- Re-suspend beads in AB
3. Add 20 μL of pre-mixed mixture consisting of 19 μL α-GFP/10 antibody + 1 μL TMR-BSA stock (6 mg/mL)
4. Let mixture sit for 5 minutes
5. Wash 10x in ABSA, re-suspending the beads in 10 μL of ABSA
Flow-through volumes are typically 10-15 μL
1. Make MB mixture, and allow this mixture to incubate for at least 15 minutes
| ABSA | 24 μL |
|---|---|
| motor dilution | 0.5 μL |
| α-GFP beads | 0.5 μL |
2. Make a flow cell with a nitrocellulose-coated coverslip
3. Flow in NaV/10 stock
4. Let cell incubate for 2 minutes
5. Wash with ABSA
6. Flow in actin dilution, typically Actin/10 or Actin/20
7. Wash with ABSA immediately
8. Flow in GO-Juice
| MB mixture | 2.5 μL |
|---|---|
| 50x GOC stock | 1 μL |
| 50x Glu/B stock | 1 μL |
| 100x CPK stock | 0.5 μL |
| 100x PCr stock | 0.5 μL |
| 50 ATP stock | 1 μL |
| AB | 43.5 μL |
