Difference between revisions of "Altman:Protocols/Single Molecule Assays/single bead"

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'' Flow-through volumes are typically 10-15 μL''
 
'' Flow-through volumes are typically 10-15 μL''
  
1. Make MB mixture:
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1. Make MB mixture, and allow this mixture to incubate for at least 15 minutes
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</table>
 
</table>
- Allow this mixture to incubate for ~15 minutes
 
  
 
2.  Make a flow cell with a nitrocellulose-coated coverslip
 
2.  Make a flow cell with a nitrocellulose-coated coverslip
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5.  Wash with ABSA
 
5.  Wash with ABSA
  
6.  Flow in actin dilution
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6.  Flow in actin dilution, typically Actin/10 or Actin/20  
* Typically Actin/10 or Actin/20  
 
  
 
7.  Wash with ABSA immediately
 
7.  Wash with ABSA immediately

Revision as of 14:07, 15 June 2013

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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



2.5 μL MB mixture 1 μL GOC stock (50x) 1 μL glucose/β-mercaptoethanol stock (50x) 0.5 μL CPK (100x) 0.5 μL PCr (100x) 1 μL ATP dilution (50x) 43.5 μL AB





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