Mehrnoosh10th January 2020 at 11:17am
Goal:
detect high quality b-tubulin signal for these three cell lines. If possible, try probing for ORF1p.
Material:
- Cell lines: N2102EP.
- 3-plex probing chamber.
- Novus #NB600‐936 Rabbit β-tubulin , 4ul from stock.
- Clone5 llama‐ derived anti‐ORF1p nanobody ‐ His tag, 5ul from stock.
- Abmart #5130 anti-ORF1, 4ul from stock.
- Mouse anti-his NB100‐64768/F 647, 5ul from stock.
- Novus #NL004, rabbit Igg, 4ul from stock.
- Novus #NL008, mouse Igg, 4ul from stock.
Protocol:
3-plex probing chamber
The following 1° antibodies / chips will be used:
| Culture/ plasmid | Supplier # | Organism | Target | Storage (C) | [Stock] (mg/ml) | [Final] (mg/ml) | Vol from Stock (ul) | Final vol (ul) | Chip # | Run Time (sec) | Chip Position |
|---|---|---|---|---|---|---|---|---|---|---|---|
| N2102E/2A6 | clone5 llama‐nanobody ‐His tag | 4 | 0.85 | 0.02 | 5 | 40 | 17630 | 90 | A | ||
| N2102E/2A6 | Novus #NB600-936 | rabbit | beta tubulin | -20 | 1.0 | 0.1 | 4 | 40 | 17630 | NA | B |
| N2102E/2A6 | Abmart #5130 (custom); same as Millipore #MABC1152 | mouse | ORF1p | 4 | 2.0 | 0.1 | 4 | 40 | 17630 | 90 | C |
The following 2° antibodies / chips will be used:
| Culture/ plasmid | Supplier # | Organism | Detection | Target | Storage (C) | [Stock] (mg/ml) | [Final] (mg/ml) | Vol from Stock (ul) | Final vol (ul) | Chip # | Run Time (sec) | Chip Position |
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| N2102E/2A6 | NB100‐ 64768AF 647 | Mouse | 647 | His | 4 | 0.82 | 0.1 | 5 | 40 | 17630 | 90 | A |
| N2102E/2A6 | Novus #NB600-936 | rabbit | 557 | beta tubulin | -20 | 1.0 | 0.1 | 4 | 40 | 17630 | NA | B |
| N2102E/2A6 | Abmart #5130 (custom); same as Millipore #MABC1152 | mouse | 637 | ORF1p | 4 | 1.0 | 0.1 | 4 | 40 | 17630 | 90 | C |
- Rehydrate chips for 10 minutes.
- Count cells, 100,000 cells/ml, 2 tubes 1 ml each, final combine all cells in 1 ml.
- Load cell on the chip.
- Setting time 8 minutes
- Run Milo - electrophoresis run time 90 sec
- Wash chips for 2x10 minutes
- Prepare 1st antibodies
- 1st antibody probing with the order shown in the picture for 2 hours.
- Wash 3 x10 minutes.
- Spin the chip for 3-5 seconds.
- Prepare 2nd antibodies.
- Spin the chip for 3-5 seconds.
- 2nd antibodies probing for an hour.
- Wash 3 x15 minutes.
- Spin the chip for 3 minutes till it's dry.
- Scan chips.
Raw files are available here
Data Analysis:
Stripping protocol:
62.5 mM Tris-HCl (pH 6.8) + 2% (w/v) SDS (20 g in 1 L) 0.8% (v/v) β-ME (14.3 M)
- Mix 62.5 mM Tris-HCl (pH 6.8) + 2% (w/v) SDS (20 g in 1 L) to make stripping buffer.
- Add 0.8% (v/v) β-ME (14.3 M) to the stripping buffer whenever you want tro strip a chip.
- Filling tube with 40mL of stripping buffer + 320 ul B-ME
- Leaving the chip inside a tube in water bath with 60 degree for 90 minutes
- 4 x 15 mins wash