Altman:Protocols/Single Molecule Assays/single bead

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* Re-suspend beads in AB
* Re-suspend beads in AB
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3. Add 20 μL of pre-mixed mixture consisting of:
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3. Add 20 μL of pre-mixed mixture consisting of 19 μL α-GFP/10 antibody + 1 μL TMR-BSA stock (6 mg/mL)
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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
4. Let mixture sit for 5 minutes
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 +
'' Flow-through volumes are typically 10-15 μL''
 +
1. Make MB mixture:
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1. Make a flow cell with a nitrocellulose-coated coverslip
 
-
2. Flow in α-GFP/10
+
<table id="Table 1" border="1" cellpadding="3" cellspacing="0">
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3. Incubate 2 minutes
 
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4. Flow in ABSA
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<tr>
 +
<th><tt> ABSA </tt></th>
 +
<th><tt> 24 μL  </tt></th>
 +
</tr>
 +
 
 +
<tr>
 +
<th><tt> motor dilution </tt></th>
 +
<th><tt> 0.5 μL </tt></th>
 +
</tr>
 +
 
 +
<tr>
 +
<th><tt> α-GFP beads </tt></th>
 +
<th><tt> 0.5 μL </tt></th>
 +
</tr>
 +
 
 +
 
 +
</table>
 +
* Allow this mixture to incubate for ~15 minutes
 +
 
 +
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
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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
 
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11. Flow in GO-Juice
 

Revision as of 17:03, 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


Flow-through volumes are typically 10-15 μL

1. Make MB mixture:


ABSA 24 μL
motor dilution 0.5 μL
α-GFP beads 0.5 μL
  • Allow this mixture to incubate for ~15 minutes

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


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