Altman:Protocols/Protein Purification/SF9 purification
From OpenWetWare
(Difference between revisions)
| Line 1: | Line 1: | ||
| + | {{Template:Altman}} | ||
| + | |||
| + | <div style="padding: 10px; width: 700px; border: 5px solid #B22222;"> | ||
| + | |||
| + | ==Small-scale purification of FLAG-tagged proteins from SF9 cells== | ||
| Line 18: | Line 23: | ||
:2 mM ATP | :2 mM ATP | ||
| - | |||
| - | |||
| - | |||
| - | |||
| - | |||
FINAL CONCENTRATION of 50x GOC stock: 10.8 mg/mL glucose oxidase, 1.8 mg/mL catalase | FINAL CONCENTRATION of 50x GOC stock: 10.8 mg/mL glucose oxidase, 1.8 mg/mL catalase | ||
Revision as of 13:02, 19 February 2013
Department of Physics, Willamette University
Small-scale purification of FLAG-tagged proteins from SF9 cells
Prepare the following beforehand:
- LYSIS BUFFER
Final concentrations:
- 200 mM NaCl
- 4 mM MgCl2
- 20 mM Imidazole, pH 7.5
- 0.5 mM EDTA
- 1 mM EGTA
- 0.5% Igepal
- 7% sucrose
- 1 mM PMSF (0.25 M stock)
- 10 μg/mL Aprotinin
- 10 μg/mL Leupeptin
- 5 mM DTT
- 2 mM ATP
FINAL CONCENTRATION of 50x GOC stock: 10.8 mg/mL glucose oxidase, 1.8 mg/mL catalase
in 1x AB-DTT and 50% Glycerol
- Glucose Oxidase, Aspergillus niger (Calbiochem, 346385)
- Catalase, from Bovine Liver (Sigma, 19.9 mg/mL)
- Glycerol, Anhydrous
1. Make 2x Assay Buffer (2xAB)
| 1 mL 2xAB | |
|---|---|
| 10x AB stock | 200 uL |
| 100x DTT stock | 20 uL |
| ddwater | 780 uL |
2. Make 43 mg/mL glucose oxidase (GO) in 2xAB.
- Combine 17 mg of GO with 400 μL 2xAB to make ~400 μL of GO.
3. Make 7.2 mg/mL catalase.
- Combine 145 μL of catalase with 255 μL of 2xAB to make 400 μL of catalase.
4. Add glycerol to the GO and catalase stocks 1:1 volume/volume.
- Add 400 μL of glycerol to each, being careful to not introduce bubbles, especially in the GO.
- This produces 100x stocks of GO and catalase.
5. Combine the GO stock and catalase stock 1:1 volume/volume to yield a 50x stock of GOC.
6. Aliquot into 50 μL stocks, which are stored at 20°C
