Altman:Protocols/Single Molecule Assays/single bead: Difference between revisions
David Altman (talk | contribs) No edit summary |
David Altman (talk | contribs) No edit summary |
||
Line 29: | Line 29: | ||
'' 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 | ||
Line 52: | Line 53: | ||
</table> | </table> | ||
2. Make a flow cell with a nitrocellulose-coated coverslip | 2. Make a flow cell with a nitrocellulose-coated coverslip | ||
Line 62: | Line 62: | ||
5. Wash with ABSA | 5. Wash with ABSA | ||
6. Flow in actin dilution | 6. Flow in actin dilution, typically Actin/10 or Actin/20 | ||
7. Wash with ABSA immediately | 7. Wash with ABSA immediately |
Revision as of 14:07, 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, 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
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 |