BL21(DE3) cells expressing His6-N-WASP were collected by centrifugation and lysed by cell disruption (Emulsiflex-C5, Avestin) in 20 mM imidazole (pH 7.0), 300 mM KCl, 5 mM βME, 0.01% NP-40, 1 mM PMSF, 100 μM antipain, 1 mM benzamidine, 100 μM leupeptin, and 1 μM pepstatin. Proteins were affinity-purified with NiNTA agarose (Qiagen). The eluate was further purified over a Source 15 Q column (Cytiva). The His6-tag was removed by TEV protease treatment at 4 °C for 16 hours. Cleaved N-WASP was then applied to a Source 15 S column (Cytiva). Fractions containing N-WASP were concentrated using Amicon Ultra Centrifugal Filter units (Millipore) and further purified by size exclusion chromatography using a Superdex 200 prepgrade column (Cytiva) in 25 mM HEPES (pH 7.5), 150 mM KCl, 1 mM DTT, and 10% glycerol.
Superdex 200 prepgrade column
Manufactured by Cytiva
The Superdex 200 prepgrade column is a size-exclusion chromatography column designed for the separation and purification of proteins, peptides, and other biomolecules. The column features a dextran-based matrix and is suitable for preparative-scale purifications.
Automatically generated - may contain errors
2 protocols using superdex 200 prepgrade column
1
Purification of Recombinant N-WASP
2
Purification of SUMO Domain Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
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All SUMO domain proteins were expressed with
an N-terminal His8-tag in BL21(DE3) T1R cells in TB media,
collected by centrifugation,
and lysed using cell disruption (Emulsiflex-C5, Avestin) in a buffer
containing 20 mM Tris-HCl (pH 8.0), 300 mM NaCl, 1 mM PMSF, 1 μg/mL
antipain, 1 μg/mL pepstatin, and 1 μg/mL leupeptin. The
lysate was clarified by centrifugation, and the supernatant was applied
to Ni-NTA agarose resin (Qiagen). The resin was first washed with
a buffer of 20 mM Tris-HCl, 300 mM NaCl, and 20 mM imidazole (pH 8.0)
and with a second wash with a buffer of 20 mM Tris-HCl, 150 mM NaCl,
and 30 mM imidazole (pH 8.0). Protein was eluted in 20 mM Tris-HCl,
150 mM NaCl, and 250 mM imidazole (pH 8.0). The His8-tag
was removed using TEV protease at 4 °C overnight. The cleaved
protein was applied to a Source15Q (Cytiva Life Sciences) anion exchange
column and eluted using a gradient from 0 to 500 mM NaCl in 20 mM
Tris-HCl (pH 8.0), 1 mM EDTA, and 1 mM DTT. Fractions containing SUMO
were pooled and further purified using a Superdex200 prepgrade column
(Cytiva Life Sciences) in 20 mM HEPES, 150 mM KCl, 1 mM MgCl2, 1 mM DTT, and 1 mM EGTA (pH 7.0).
an N-terminal His8-tag in BL21(DE3) T1R cells in TB media,
collected by centrifugation,
and lysed using cell disruption (Emulsiflex-C5, Avestin) in a buffer
containing 20 mM Tris-HCl (pH 8.0), 300 mM NaCl, 1 mM PMSF, 1 μg/mL
antipain, 1 μg/mL pepstatin, and 1 μg/mL leupeptin. The
lysate was clarified by centrifugation, and the supernatant was applied
to Ni-NTA agarose resin (Qiagen). The resin was first washed with
a buffer of 20 mM Tris-HCl, 300 mM NaCl, and 20 mM imidazole (pH 8.0)
and with a second wash with a buffer of 20 mM Tris-HCl, 150 mM NaCl,
and 30 mM imidazole (pH 8.0). Protein was eluted in 20 mM Tris-HCl,
150 mM NaCl, and 250 mM imidazole (pH 8.0). The His8-tag
was removed using TEV protease at 4 °C overnight. The cleaved
protein was applied to a Source15Q (Cytiva Life Sciences) anion exchange
column and eluted using a gradient from 0 to 500 mM NaCl in 20 mM
Tris-HCl (pH 8.0), 1 mM EDTA, and 1 mM DTT. Fractions containing SUMO
were pooled and further purified using a Superdex200 prepgrade column
(Cytiva Life Sciences) in 20 mM HEPES, 150 mM KCl, 1 mM MgCl2, 1 mM DTT, and 1 mM EGTA (pH 7.0).
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