Altman:Protocols/Single Molecule Assays/single bead

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Department of Physics, Willamette University


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Single Bead Optical Trapping Assay

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