Altman:Protocols/Protein Purification/SF9 purification

Department of Physics, Willamette University

'

Home        Contact        Lab Members        Publications        Research        Protocols        Pictures       

Small-scale purification of FLAG-tagged proteins from SF9 cells

Prepare the following beforehand:

• LYSIS BUFFER
  • NOTE: if you are freezing down cells before the purification, leave out the Igepal and Sucrose. This will require you to make 2x Lysis Buffer without these two ingredients.

Final concentrations:

200 mM NaCl

4 mM MgCl2

20 mM Imidazole, pH 7.5

0.5 mM EDTA

1 mM EGTA

0.5% (v/v) Igepal

7% (w/v) Sucrose

1 mM PMSF (0.25 M stock)

10 μg/mL Aprotinin

10 μg/mL Leupeptin

5 mM DTT

2 mM ATP

	5 mL total
2x lysis buffer	2.5 mL
1 mg/mL aprotinin stock	50 uL
1 mg/mL leupeptin stock	50 uL
1 M DTT	25 uL
100 mM ATP	100 uL
0.25 M PMSF	20 uL
diwater	2.255 mL

• 2x LYSIS BUFFER

Final Concentrations

400 mM NaCl

8 mM MgCl2

40 mM Imidazole, pH 7.5

1 mM EDTA

2 mM EGTA

1% (v/v) Igepal

14% (w/v) sucrose

	20 mL total
5 M NaCl	1.6 mL
1 M MgCl2	160 uL
I M Imidazole, pH 7.5	800 uL
0.5 M EDTA	40 uL
0.5 M EGTA	80 uL
Igepal	200 uL
Sucrose	2.8 g
diwater	raise to 20 mL