Altman:Protocols/Single Molecule Assays/single bead

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

1. Make a flow cell with a nitrocellulose-coated coverslip

2. Flow in α-GFP/10

3. Incubate 2 minutes

4. Flow in ABSA

5. Incubate 2 minutes

6. Flow in motor dilution

7. Incubate 2 minutes

8. Flow in ABSA

9. Flow in Actin dilution

10. Incubate 2 minutes

11. Flow in GO-Juice

RECIPES

Buffer #1

	50 uL Buffer #1
50x GOC stock	2 μL
100x CPK stock	1 μL
100x ATP stock	1 μL
AB	46 μL

Buffer #2

	50 μL Buffer #2
50x Glu/B stock	2 μL
100x PCr stock	1 μL
AB	47 μL

GO-Juice

	20 μL GO-Juice
Buffer #1	10 μL
Buffer #2	10 μL