Clarified and filtered culture media were applied to HisTrap Excel 5 ml column (Cytiva) at 3 ml/min followed by 10 CV wash with 50 mM sodium phosphate, 300 mM NaCl, 10 mM imidazole, pH 8.0. Bound proteins were eluted with 50 mM sodium phosphate, 300 mM NaCl, 500 mM imidazole, pH 8.0. Pooled eluate was loaded onto Strep-Tactin Superflow Plus cartridge (1 ml or 5 ml at 0.5 ml/min or 3 ml/min; Qiagen). The resin was washed with 10 CV of 50 mM sodium phosphate, 300 mM NaCl, pH 8.0 and proteins were eluted in the same buffer with 2.5 mM desthiobiotin. Fractions containing protein were pooled, concentrated in 30 kDa MWCO concentrator (Pierce) to 0.1-0.4 ml and manually injected onto Superose 6 Increase (Cytiva) equilibrated in 20 mM Hepes, 150 mM NaCl, pH 7.6. Protein concentration was determined using Nanodrop 2000 or 8000 (Thermo Fisher).
Strep tactin superflow plus cartridge
Manufactured by Qiagen
Sourced in Germany
The Strep-Tactin Superflow Plus cartridge is a lab equipment product designed for the purification of Strep-tagged proteins. It provides a high-capacity matrix for efficient capture and elution of Strep-tagged proteins.
Automatically generated - may contain errors
Lab products found in correlation
Nanodrop 2000, by Thermo Fisher Scientific (2 mentions)
Histrap excel 5 ml column, by Cytiva (1 mentions)
Superose 6 increase, by Cytiva (1 mentions)
Thrombin, by Cytiva (1 mentions)
Superdex 200 10 300 gl column, by Cytiva (1 mentions)
Thrombin, by Merck Group (1 mentions)
Total protein kit, by Merck Group (1 mentions)
6 protocols using strep tactin superflow plus cartridge
1
Purification of His-tagged Proteins
2
Purification of Recombinant Proteins
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Cells were lysed by incubating at room temperature for 1 h and sonicating four times for 15 s at 20% amplitude using a Branson Digital Sonifier Model 250-D (Emerson, Ferguson, MO, USA). After centrifugation (30 min, 40,000 × g, 4 °C), the proteins were recovered from the supernatant by Strep-Tactin affinity chromatography (Strep-Tactin® Superflow Plus Cartridge, Qiagen Hilden, Germany). The column was washed with 20 mM TEA containing 400 mM NaCl (pH 8.0), and proteins were eluted using 2.5 mM D-desthiobiotin in the same buffer. Purified enzymes were re-buffered in 20 mM TEA (pH 8.0) and stored at 4 °C in 10% (v/v) glycerol47 (link).
3
Recombinant Ubiquitin Purification
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The gene encoding human Ub with an N-terminal Strep-tag II was inserted into pGEX-2TK where the GST-coding sequence was removed. The plasmid was transformed into E. coli BL21 (DE3). For expression of Ub, cells were cultured in LB medium at 37 °C. At OD600 = 0.6–0.8, 1 mM IPTG was added, and after 8 h cells were harvested by centrifugation.
Cells were resuspended in 100 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, 0.1 mg/ml lysozyme, 0.01% (v/v) Triton X-100, incubated on ice and lysed by sonication. After centrifugation, the supernatant was pre-cleared by heat denaturation. Therefore, the mixture was incubated at 60 °C for 20 min, followed by centrifugation. The resulting supernatant was manually loaded onto a Strep-Tactin Superflow Plus Cartridge (Qiagen), washed with 100 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1 mM EDTA, followed by elution with 2.5 mM desthiobiotin. Elution fractions were concentrated by ultrafiltration and the concentration determined by BCA assay/SDS-PAGE and Coomassie blue staining.
Cells were resuspended in 100 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, 0.1 mg/ml lysozyme, 0.01% (v/v) Triton X-100, incubated on ice and lysed by sonication. After centrifugation, the supernatant was pre-cleared by heat denaturation. Therefore, the mixture was incubated at 60 °C for 20 min, followed by centrifugation. The resulting supernatant was manually loaded onto a Strep-Tactin Superflow Plus Cartridge (Qiagen), washed with 100 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1 mM EDTA, followed by elution with 2.5 mM desthiobiotin. Elution fractions were concentrated by ultrafiltration and the concentration determined by BCA assay/SDS-PAGE and Coomassie blue staining.
4
Affinity Purification of Netrin-1ΔC
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Netrin-1ΔC was purified by affinity chromatography using a 5 mL Strep-tactin Superflow Plus cartridge (Qiagen) equilibrated with 50 mM Tris, pH 8, and 500 mM NaCl. Each ECS collection was loaded onto the column and washed with 3 column volumes of 50 mM Tris, pH 8, 500 mM NaCl followed by 5 column volumes of 50 mM Tris, pH 8, and 1 M NaCl to eliminate non-specific binding. Protein elution was performed with 2.5 mM of d-Desthiobiotin (MilliporeSigma) in 50 mM Tris, pH 8, and 0.5 M NaCl. The Strep-tag was removed with 1 unit of Thrombin (MilliporeSigma) per mg of protein in a dialysis bag with 25 kDa molecular weight cut-off (Repligen, Spectra/Por) in 50 mM Tris, pH 8, 1 M NaCl, 0.15 M glycine, and 2.5 mM CaCl2 and incubated overnight at room temperature. Finally, Thrombin was removed using a Benzamidine column (HiTrap® Benzamidine FF, Cytiva) following the manufacturer’s protocol. Tag-free protein was analyzed and purified using an ÄKTA FPLC system with a Superdex 200 10/300 GL column (Cytiva, Healthcare), pre-equilibrated with 50 mM Tris, pH 7.5, and 1 M NaCl. Purified Netrin-1ΔC was concentrated to 1 mg/mL (using molecular weight of 49.5 kDa and extinction coefficient of 49455 M−1 cm−1 obtained from ProtParam (ExPASy Server)) and stored at 4 °C until further use.
5
Purification of FRET Mutant Variants
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The FRET mutant variants—each of the two parallels (25 mg)—were lysed in Bug Buster Protein Extraction Reagent (Novagen, Darmstadt, Germany) detergent, cooled on ice, and centrifuged, and the cleared lysates were loaded on a 1 mL Strep-Tactin Superflow Plus Cartridge (QIAGEN, Hilden, Germany). According to the QIAGEN protocol, the proteins were washed and isolated in a volume of 4 mL of Strep-Tactin elution buffer (pH 8.0). The concentration of proteins was assayed using a Total Protein Kit, Micro Lowry, Onishi & Barr Modification (Sigma-Aldrich, St. Louis, MO, USA). The elution products were kept at −30 °C until further use.
6
Purification of NET-1 and UNC-5B with Thrombin Cleavage
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Gallus gallus NET-1 with truncated C-terminal domain (Uniprot ID: Q90922, amino acid [aa] 26-458), and UNC-5B (Uniprot ID: Q8K1S3, aa 25-300) all containing a thrombin cleavable C-terminal double Strep II ® -tag were expressed in human embryonic kidney 293 T cells using the Sleeping beauty transposon system (Kowarz et al. 2015) (link). Confluent cells were induced with 0.5 μg/mL Doxycycline in Dulbecco's Modified Eagle's Media and 2.5% fetal bovine serum. Collected media was passed through a Strep-Tactin Superflow Plus cartridge (Qiagen Inc., Toronto, Canada) to affinity purify the proteins. Subsequently, the affinity tag was removed by thrombin digestion during dialysis. The receptor UNC-5B was dialyzed into 50 mM Tris pH 7.5, 200 mM NaCl and NET-1 was dialyzed into 50 mM Tris pH 7.5, 500 mM NaCl, 500 mM (NH 4 ) 2 SO 4 to remove bound heparins. Final purification was performed on a Superdex 200 with the appropriate dialysis buffer.
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