Altman:Protocols/Single Molecule Assays/single bead

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(New page: {{Template:Altman}} <div style="padding: 10px; width: 700px; border: 5px solid #B22222;"> ==Single Bead Optical Trapping Assay== ''Before you start'' * Prepare an aliquot of α-GFP/10 (...)
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''Make α-GFP beads''
''Make α-GFP beads''
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1. Take 10 μL of 1-μm diameter carboxylated beads
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 +
2. Wash beads 3x in AB
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''All wash steps should be performed as follows''
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* Spin down beads in a benchtop centrifuge at max speed (14k rpm) for 1-2 minutes
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* Carefully, pull off the supernatant
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* Re-suspend beads in AB
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3. Add 20 μL of pre-mixed mixture consisting of:
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      19 μL α-GFP/10 antibody
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      1 μL TMR-BSA stock (6 mg/mL)
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4. Let mixture sit for 5 minutes
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5. Wash 10x in ABSA, re-suspending the beads in 10 μL of ABSA
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 +

Revision as of 16:58, 15 June 2013


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


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