Altman:Protocols/Single Molecule Assays/single bead: Difference between revisions
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* Re-suspend beads in AB | * Re-suspend beads in AB | ||
3. Add 20 μL of pre-mixed mixture consisting of | 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 | 4. Let mixture sit for 5 minutes | ||
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'' Flow-through volumes are typically 10-15 μL'' | |||
1. Make MB mixture: | |||
<table id="Table 1" border="1" cellpadding="3" cellspacing="0"> | |||
4. Flow in ABSA | <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 | |||
Revision as of 14:03, 15 June 2013
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 #150 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 |