Altman:Protocols/Protein Purification/SF9 purification

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<table id="Table 1" border="1" cellpadding="3" cellspacing="0">
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 +
 +
* 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
 +
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Revision as of 12:07, 19 February 2013


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% (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
  • 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
5 mL total
2x lysis buffer 2.5 mL
1 mg/mL aprotinin stcok 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
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