Cells were lysed in “His-purification buffer” [50 mM tris (pH 7.5), 300 mM NaCl, 1.5 mM MgCl2, and 0.15% CA-630 (Sigma-Aldrich)] with the Protease Inhibitor Mix HP (Serva), and the cleared lysate was incubated with a Ni Sepharose 6 Fast Flow resin (GE Healthcare). The unbound material was washed out with His-purification buffer in the Poly-Prep Chromatography Column (Bio-Rad), and proteins were eluted with a “His-elution buffer” [50 mM tris (pH 7.5), 150 mM NaCl, 1.5 mM MgCl2, and 500 mM imidazole]. The eluate was incubated with a StrepTactin Sepharose High Performance resin (GE Healthcare), unbound material was washed out on the Poly-Prep Chromatography Column (Bio-Rad) with “Strep-purification buffer” [50 mM tris (pH 7.5), 300 mM NaCl, and 0.5 mM dithiothreitol (DTT)], and proteins were eluted with “Strep-elution buffer” [50 mM tris (pH 7.5), 150 mM NaCl, 0.5 mM DTT, and 5 mM biotin].
Streptactin sepharose high performance resin
Manufactured by GE Healthcare
StrepTactin Sepharose High Performance resin is a chromatography resin designed for the purification of Strep-tag fusion proteins. It is composed of Sepharose beads with covalently coupled Strep-Tactin, a modified streptavidin protein that binds to the Strep-tag sequence with high affinity. This resin can be used for the efficient capture and purification of Strep-tag labeled proteins from complex samples.
Automatically generated - may contain errors
Lab products found in correlation
Nupage bis tris gel, by Thermo Fisher Scientific (2 mentions)
Ca 630, by Merck Group (1 mentions)
Protease inhibitor mix hp, by Serva Electrophoresis (1 mentions)
Ni sepharose 6 fast flow resin, by GE Healthcare (1 mentions)
Poly prep chromatography column, by Bio-Rad (1 mentions)
S1 rbd, by Sino Biological (1 mentions)
Dh10bac competent cells, by Thermo Fisher Scientific (1 mentions)
Hitrap protein g hp column, by GE Healthcare (1 mentions)
Histrap hp column, by GE Healthcare (1 mentions)
Sf9 cells, by American Type Culture Collection (1 mentions)
5 protocols using streptactin sepharose high performance resin
1
Tandem Affinity Purification of Proteins
2
FANCD2-FANCI Interaction Assay
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We used purified StrepII-tagged FANCD2 and His-tagged FANCI to probe
their interaction. In a total volume of 50 μl per reaction, FANCD2:FANCI
at molar ratios 1:0, 1:0.5, 1:1, 1:2, 1:5 and 0:5 were mixed in 50 mM HEPES pH
7.5, 150 mM NaCl and 1 mM TCEP and incubated for 15 min at room temperature.
Each reaction was applied to 20 μl of StrepTactin Sepharose High
Performance resin (GE Healthcare Life Sciences) equilibrated in the same buffer.
The loaded resin was then incubated for 30 min at 4 °C. The unbound
fraction was removed and the resin further washed twice using 250 μl of
the same buffer. The bound fraction was analyzed by SDS-PAGE using 4–12%
NuPAGE Bis-Tris gels (ThermoFisher Scientific).
their interaction. In a total volume of 50 μl per reaction, FANCD2:FANCI
at molar ratios 1:0, 1:0.5, 1:1, 1:2, 1:5 and 0:5 were mixed in 50 mM HEPES pH
7.5, 150 mM NaCl and 1 mM TCEP and incubated for 15 min at room temperature.
Each reaction was applied to 20 μl of StrepTactin Sepharose High
Performance resin (GE Healthcare Life Sciences) equilibrated in the same buffer.
The loaded resin was then incubated for 30 min at 4 °C. The unbound
fraction was removed and the resin further washed twice using 250 μl of
the same buffer. The bound fraction was analyzed by SDS-PAGE using 4–12%
NuPAGE Bis-Tris gels (ThermoFisher Scientific).
3
Spike Protein Expression and Purification
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The spike protein ectodomain was expressed and purified using a previously described protocol (21 (link), 29 (link)). Briefly, the construct was transformed into bacterial DH10Bac competent cells (Invitrogen); then, the extracted bacmid was transfected into Sf9 cells (American Type Culture Collection). The supernatant of the cell culture containing the secreted S glycoprotein was harvested at 60 h after infection and concentrated, and the buffer was changed to binding buffer (10 mM HEPES, pH 7.2, and 500 mM NaCl). The S-trimer (HCoV-229E and SARS-CoV) including Strep tag was captured using StrepTactin Sepharose high-performance resin (GE Healthcare) (21 (link)). HCoV-229E S1, S1-NTD, S1-RBD, and SARS-CoV S1-RBD including FC tag were harvested using a HiTrap Protein G HP column (GE Healthcare, Uppsala, Sweden). Besides, for SARS-CoV-2, the S-trimer (item no. 40589-V08B1) and S1-RBD (item no. 40592-V05H) were purchased from Sino Biological, Inc. In addition, the hAPN and hACE2 including His tag were harvested using HisTrap HP columns (GE Healthcare, Uppsala, Sweden). Finally, the harvested proteins were equilibrated with buffer (10 mM HEPES, pH 7.2, and 150 mM NaCl).
4
FANCD2-FANCI Interaction Assay
5
Smc5/6 Hexamer Interaction Analysis
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For interaction analyses between Nse5/6 complexes with Twin‐Strep‐tagged Smc5/6 hexamer, the Nse5/6 complexes (or Nse6N‐CPD‐His) were diluted to 1 µM in a total volume of 250 µl with buffer (10 mM Hepes‐KOH pH 7.5, 150 mM potassium acetate, 2 mM MgCl2, 20% glycerol) and an input sample was removed and boiled for 5 min after mixing with 2 × SDS gel‐loading dye. StrepTactin Sepharose High Performance resin (GE Healthcare; 20 µl resin per pulldown) was incubated either with only 250 µl buffer or with the same buffer containing 0.5 µM Twin‐Strep‐tagged Smc5/6 hexamer. After 1‐h incubation at 4°C on a rotating wheel, the resin was collected by centrifugation (2 min at 700 g) and washed twice with 500 µl of buffer to remove unbound hexamer. Pre‐diluted Nse5/6 complexes were then incubated with either empty or loaded resin for 1 h at 4°C on a rotating wheel, and afterwards, the resin was collected by centrifugation and washed twice with 1 ml of buffer. Bound material was eluted with buffer supplemented with 2.5 mM desthiobiotin and analysed by SDS–PAGE.
Pulldowns between Smc6Δhinge(EQ)‐3C‐Twin‐Strep / Nse4(N) and Smc5Δhinge(EQ) / Nse2 / Nse4can were carried out in a similar way, except that the indicated nucleotides were added to 2 mM final concentration during binding and washing.
Pulldowns between Smc6Δhinge(EQ)‐3C‐Twin‐Strep / Nse4(N) and Smc5Δhinge(EQ) / Nse2 / Nse4can were carried out in a similar way, except that the indicated nucleotides were added to 2 mM final concentration during binding and washing.
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