Altman:Protocols/Protein Purification/SF9 purification

Department of Physics, Willamette University

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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