Altman:Protocols/Single Molecule Assays/single bead: Difference between revisions
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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 | - Spin down beads in a benchtop centrifuge at max speed (14k rpm) for 1-2 minutes | ||
- Carefully, pull off the supernatant | - Carefully, pull off the supernatant | ||
- Re-suspend beads in AB | - Re-suspend beads in AB | ||
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8. Flow in GO-Juice | 8. Flow in GO-Juice | ||
<table id="Table 1" border="1" cellpadding="3" cellspacing="0"> | <table id="Table 1" border="1" cellpadding="3" cellspacing="0"> | ||
| Line 94: | Line 77: | ||
Buffer #1 | Buffer #1 | ||
<tr> | <tr> | ||
<th><tt> </tt></th> | <th><tt> MB mixture </tt></th> | ||
<th><tt> | <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> 1 μL </tt></th> | <th><tt> 1 μL </tt></th> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
<th><tt> | <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> | <th><tt> 100x CPK stock </tt></th> | ||
<th><tt> | <th><tt> 0.5 μL </tt></th> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
<th><tt> | <th><tt> 100x PCr stock </tt></th> | ||
<th><tt> | <th><tt> 0.5 μL </tt></th> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
<th><tt> | <th><tt> 50 ATP stock </tt></th> | ||
<th><tt> 1 μL </tt></th> | <th><tt> 1 μL </tt></th> | ||
</tr> | </tr> | ||
| Line 141: | Line 108: | ||
<tr> | <tr> | ||
<th><tt> AB </tt></th> | <th><tt> AB </tt></th> | ||
<th><tt> | <th><tt> 43.5 μL </tt></th> | ||
</tr> | </tr> | ||
</table> | </table> | ||
Revision as of 14:10, 15 June 2013
Department of Physics, Willamette University
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 |
