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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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==Single Bead Optical Trapping Assay==
==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''
''Before you start, prepare an aliquot of α-GFP/10 (5 μL of α-GFP 0.5 mg/mL stock + 45 μL AB)''




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


2. Flow in α-GFP/10
''Make α-GFP beads''


3. Incubate 2 minutes
1. Take 10 μL of 1-μm diameter carboxylated beads


4. Flow in ABSA
2. Wash beads 3x in AB
''All wash steps should be performed as follows''


5. Incubate 2 minutes
- Spin down beads in a benchtop centrifuge at max speed (14k rpm) for 1-2 minutes


6. Flow in motor dilution
- Carefully, pull off the supernatant


7. Incubate 2 minutes
- Re-suspend beads in AB


8. Flow in ABSA
3. Add 20 μL of pre-mixed mixture consisting of 19 μL α-GFP/10 antibody + 1 μL TMR-BSA stock (6 mg/mL)


9. Flow in Actin dilution
4. Let mixture sit for 5 minutes


10. Incubate 2 minutes
5. Wash 10x in ABSA, re-suspending the beads in 10 μL of ABSA


11. Flow in GO-Juice


'' Flow-through volumes are typically 10-15 μL''


'''RECIPES'''
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">




Buffer #1
<tr>
<tr>
<th><tt> </tt></th>
<th><tt> ABSA </tt></th>
<th><tt>50 uL Buffer #1 </tt></th>
<th><tt> 24 μL  </tt></th>
</tr>
</tr>


<tr>
<tr>
<th><tt> 50x GOC stock </tt></th>
<th><tt> motor dilution </tt></th>
<th><tt> 2 μL </tt></th>
<th><tt> 0.5 μL </tt></th>
</tr>
</tr>


<tr>
<tr>
<th><tt> 100x CPK stock </tt></th>
<th><tt> α-GFP beads </tt></th>
<th><tt> 1 μL </tt></th>
<th><tt> 0.5 μL </tt></th>
</tr>
</tr>


<tr>
</table>
<th><tt> 100x ATP stock </tt></th>
 
<th><tt> 1 μL </tt></th>
2.  Make a flow cell with a nitrocellulose-coated coverslip
</tr>
 
3.  Flow in NaV/10 stock
 
4.  Let cell incubate for 2 minutes
 
5.  Wash with ABSA


<tr>
6. Flow in actin dilution, typically Actin/10 or Actin/20
<th><tt> AB </tt></th>
<th><tt> 46 μL </tt></th>
</tr>


</table>
7.  Wash with ABSA immediately


8.  Flow in GO-Juice


Buffer #2
<table id="Table 1" border="1" cellpadding="3" cellspacing="0">
<table id="Table 1" border="1" cellpadding="3" cellspacing="0">
<tr>
<tr>
<th><tt> </tt></th>
<th><tt> MB mixture </tt></th>
<th><tt> 50 μL Buffer #2  </tt></th>
<th><tt>2.5 μL   </tt></th>
</tr>
</tr>


<tr>
<tr>
<th><tt> 50x Glu/B stock </tt></th>
<th><tt> 50x GOC stock </tt></th>
<th><tt> 2 μL </tt></th>
<th><tt> 1 μL </tt></th>
</tr>
</tr>


<tr>
<tr>
<th><tt> 100x PCr 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> 47 μL </tt></th>
<th><tt> 0.5 μL </tt></th>
</tr>
</tr>
</table>
GO-Juice
<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> 20 μL GO-Juice  </tt></th>
<th><tt> 0.5 μL </tt></th>
</tr>
</tr>


<tr>
<tr>
<th><tt> Buffer #1 </tt></th>
<th><tt> 50 ATP stock </tt></th>
<th><tt> 10 μL </tt></th>
<th><tt> 1 μL </tt></th>
</tr>
</tr>


<tr>
<tr>
<th><tt> Buffer #2 </tt></th>
<th><tt> AB </tt></th>
<th><tt> 10 μL </tt></th>
<th><tt> 43.5 μL </tt></th>
</tr>
</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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