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

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''Before you start''
''Before you start, prepare an aliquot of α-GFP/10 (5 μL of α-GFP 0.5 mg/mL stock + 45 μL AB)''
* Prepare an aliquot of α-GFP/10 (5 μL of α-GFP 0.5 mg/mL stock + 45 μL AB)




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2. Wash beads 3x in AB
2. Wash beads 3x in AB
''All wash steps should be performed as follows''
''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
- Spin down beads in a benchtop centrifuge at max speed (14k rpm) for 1-2 minutes
* Re-suspend beads in AB
 
- 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)
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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'' Flow-through volumes are typically 10-15 μL''
'' Flow-through volumes are typically 10-15 μL''


1. Make MB mixture:
1. Make MB mixture, and allow this mixture to incubate for at least 15 minutes
 


<table id="Table 1" border="1" cellpadding="3" cellspacing="0">
<table id="Table 1" border="1" cellpadding="3" cellspacing="0">
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<th><tt> 0.5 μL </tt></th>
<th><tt> 0.5 μL </tt></th>
</tr>
</tr>


</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
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


8.  Flow in GO-Juice
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'''


<table id="Table 1" border="1" cellpadding="3" cellspacing="0">
<table id="Table 1" border="1" cellpadding="3" cellspacing="0">
Buffer #1
<tr>
<tr>
<th><tt> </tt></th>
<th><tt> MB mixture </tt></th>
<th><tt>50 uL Buffer #1 </tt></th>
<th><tt>2.5 μL  </tt></th>
</tr>
</tr>


<tr>
<tr>
<th><tt> 50x GOC stock </tt></th>
<th><tt> 50x GOC stock </tt></th>
<th><tt> 2 μL </tt></th>
</tr>
<tr>
<th><tt> 100x CPK stock </tt></th>
<th><tt> 1 μL </tt></th>
<th><tt> 1 μL </tt></th>
</tr>
</tr>


<tr>
<tr>
<th><tt> 100x ATP stock </tt></th>
<th><tt> 50x Glu/B stock </tt></th>
<th><tt> 1 μL </tt></th>
<th><tt> 1 μL </tt></th>
</tr>
</tr>


<tr>
<tr>
<th><tt> AB </tt></th>
<th><tt> 100x CPK stock </tt></th>
<th><tt> 46 μL </tt></th>
<th><tt> 0.5 μL </tt></th>
</tr>
</tr>
</table>
Buffer #2
<table id="Table 1" border="1" cellpadding="3" cellspacing="0">


<tr>
<tr>
<th><tt> </tt></th>
<th><tt> 100x PCr stock </tt></th>
<th><tt> 50 μL Buffer #2  </tt></th>
<th><tt> 0.5 μL </tt></th>
</tr>
 
<tr>
<th><tt> 50x Glu/B stock </tt></th>
<th><tt> 2 μL </tt></th>
</tr>
</tr>


<tr>
<tr>
<th><tt> 100x PCr stock </tt></th>
<th><tt> 50 ATP stock </tt></th>
<th><tt> 1 μL </tt></th>
<th><tt> 1 μL </tt></th>
</tr>
</tr>
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<tr>
<tr>
<th><tt> AB </tt></th>
<th><tt> AB </tt></th>
<th><tt> 47 μL </tt></th>
<th><tt> 43.5 μL </tt></th>
</tr>
 
</table>
 
 
 
GO-Juice
<table id="Table 1" border="1" cellpadding="3" cellspacing="0">
 
<tr>
<th><tt> </tt></th>
<th><tt> 20 μL GO-Juice  </tt></th>
</tr>
 
<tr>
<th><tt> Buffer #1 </tt></th>
<th><tt> 10 μL </tt></th>
</tr>
</tr>
<tr>
<th><tt> Buffer #2 </tt></th>
<th><tt> 10 μL </tt></th>
</tr>


</table>
</table>

Latest revision as of 14:11, 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, 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


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