The pET15b expression vector encoding 3FN3 was provided to us by Dr. Tomoo Ohashi and Dr. Harold Erickson (Duke University) 12 (link), 13 (link). The His-tagged fusion protein was expressed in Escherichia coli BL21-CodonPlus(DE3)-RIPL cells (Agilent) and purified by affinity chromatography on a HisTrap FF column (GE Healthcare). The N-terminal His-tag was cleaved off with thrombin, and 3FN3 was separated from thrombin and the His-tag by affinity chromatography on HiTrap Benzamidine FF (high sub) (GE Healthcare) and HisTrap FF columns respectively. 3FN3 was then further purified by size-exclusion chromatography on Superdex 75 resin (GE Healthcare). The recombinant 3FN3 protein consisted of six extraneous amino acids at the N-terminus, residues 808-905 of human fibronectin (P02751), and two extraneous amino acids at the C-terminus (gshmgtTTAP...PRSDgt).
Histrap ff column
Manufactured by GE Healthcare
Sourced in United States, Sweden, United Kingdom, Germany, Canada
The HisTrap FF column is a chromatography column designed for the purification of histidine-tagged proteins. It utilizes a resin with high-performance agarose beads that are pre-charged with Ni²⁺ ions, enabling efficient capture and purification of the target proteins. The column is optimized for fast and effective separation, making it a reliable tool for laboratory research and protein purification applications.
Automatically generated - may contain errors
Lab products found in correlation
Hitrap desalting column, by GE Healthcare (6 mentions)
Hiload superdex g200 16 60 column, by GE Healthcare (5 mentions)
Protease inhibitor cocktail, by Roche (4 mentions)
Superdex 200 increase 10 300 gl, by GE Healthcare (4 mentions)
Hitrap q hp column, by GE Healthcare (4 mentions)
Superdex 200 increase 10 300 gl column, by GE Healthcare (4 mentions)
Expi293 expression system, by Thermo Fisher Scientific (4 mentions)
Akta fplc system, by GE Healthcare (3 mentions)
Superdex 200, by GE Healthcare (3 mentions)
Kappaselect, by GE Healthcare (3 mentions)
Pd 10 desalting column, by GE Healthcare (3 mentions)
Hiload 16 600 superdex 200 pg column, by GE Healthcare (3 mentions)
Kta pure system, by GE Healthcare (3 mentions)
Quixstand diafiltration system, by GE Healthcare (3 mentions)
Superdex 75 column, by GE Healthcare (3 mentions)
Pet sumo expression vector, by Thermo Fisher Scientific (3 mentions)
Ni affinity chromatography, by GenScript (2 mentions)
Hitrap protein a hp, by GE Healthcare (2 mentions)
Hiload 16 60 superdex 75, by GE Healthcare (2 mentions)
Akta prime plus fplc system, by GE Healthcare (2 mentions)
217 protocols using histrap ff column
1
Expression and Purification of 3FN3 Fibronectin Domain
2
Purification of MaeR and MaeK Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
All proteins with the exception of MaeR full length protein and MaeRREC were expressed in the BL21-codonplus(DE3)-RIL strain using 1 mM IPTG at 20 °C overnight. Proteins were purified by affinity chromatography in a 5-ml HisTrap FF column (GE Healthcare) equilibrated with buffer A (20 mM Tris-HCl pH 7.5, 20 mM (NH4)2SO4, 500 mM NaCl), washed with buffer A plus 30 mM of imidazol and eluted with buffer A plus 300 mM of imidazol. MaeR full length protein and the MaeRREC domain protein were expressed in DH5α strain using 1 mM IPTG at 20 °C overnight. The purification of proteins was performed using 1 ml HisTrap FF columns (GE Healthcare) equilibrated with buffer C (20 mM Bis-Tris pH 6, 20 mM (NH4)2SO4, 500 mM NaCl, 10%glycerol), washed with buffer C plus 30 mM of imidazol and eluted with buffer C plus 300 mM of imidazol. The MaeR mutants used (D54A and D54N) were expressed and purified as wild type MaeR proteins. All the proteins used for in vitro assays were loaded in a gel filtration column, Superdex75 16/60 (GeHealthcare) for MaeR proteins and Superdex200 16/60 (GE Healthcare) for MaeKC protein. The proteins were concentrated around 10 mgml−1 with a 10 K Amicon Ultra Centrifuge Filters (Millipore) and stored at −80 °C.
3
Expression and Purification of CTX-M-14 Mutants
4
Purification of recombinant TUT4 and Lin28b proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Recombinant TUT7 951–1,495 (rTUT7) and His-rTUT7 were prepared as previously described (Lim et al, 2014 (link)). For purification of recombinant TUT4 protein, human TUT4 267–1,312 was inserted into a self-modified pMAL expression vector, which fuses a hexa-His tag plus a maltose-binding protein tag at the N-terminus to the target protein. The plasmid was transformed into E. coli BL21(DE3)-RIL strain (Stratagene). The cells were cultured at 37°C until OD600 reached 1.0, and then the protein expression was induced with 1 mM IPTG at 16°C overnight. The hexa-His-MBP tagged protein was purified using a HisTrap FF column (GE Healthcare). The tag was cleaved by TEV protease and further removed by a second step HisTrap FF column (GE Healthcare) purification. The target protein was further purified by a Heparin FF column (GE Healthcare) and a Hiload Superdex G200 16/60 column (GE Healthcare). Recombinant Lin28b was prepared as previously described (Yeom et al, 2011 (link)).
5
Purification and Crystallization of E. coli RppH
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For the in vitro decapping assays, E. coli RppH and RppH-E57A, each bearing an amino-terminal hexahistidine tag, were produced in E. coli from plasmids pPlacRppH6 and pPlacRppH6-E57A, respectively, purified by affinity chromatography on TALON beads, and stored at −80 °C in a buffer containing HEPES, pH 7.5 (10 mM), NaCl (300 mM), and glycerol (50% vol/vol) (3 (link), 14 (link)).
Structure determination was performed with a truncated variant of E. coli RppH that bore an additional serine residue at the amino terminus and lacked carboxyl-terminal residues 159 through 176, which were replaced by two alanines (16 (link), 18 (link)). This variant, RppHt, retained enzymatic activity but yielded better crystals than the full-length protein. It was produced as a fusion with amino-terminal tandem decahistidine and SUMO tags by using a T7 RNA polymerase–based expression system in E. coli strain BL21(DE3). The recombinant protein was purified by affinity chromatography on a HisTrap FF column (GE Healthcare), and the tags were cleaved off by a His-tagged variant of ULP1 protease, leaving an extra serine residue at the amino terminus. The affinity tags and the protease were removed by passage through a HisTrap FF column, and RppHt was further purified by ion-exchange chromatography on a HiTrap SP column and gel filtration on Superdex 75 (GE Healthcare).
Structure determination was performed with a truncated variant of E. coli RppH that bore an additional serine residue at the amino terminus and lacked carboxyl-terminal residues 159 through 176, which were replaced by two alanines (16 (link), 18 (link)). This variant, RppHt, retained enzymatic activity but yielded better crystals than the full-length protein. It was produced as a fusion with amino-terminal tandem decahistidine and SUMO tags by using a T7 RNA polymerase–based expression system in E. coli strain BL21(DE3). The recombinant protein was purified by affinity chromatography on a HisTrap FF column (GE Healthcare), and the tags were cleaved off by a His-tagged variant of ULP1 protease, leaving an extra serine residue at the amino terminus. The affinity tags and the protease were removed by passage through a HisTrap FF column, and RppHt was further purified by ion-exchange chromatography on a HiTrap SP column and gel filtration on Superdex 75 (GE Healthcare).
6
Rhino Chromodomain Protein Purification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The D. melanogaster Rhino chromodomain (19–85) was inserted into the pETSumo expression vector (Invitrogen), which fuses a hexa-His tag plus a yeast sumo tag at the N terminus to the target protein. The plasmid was transformed into the Escherichia coli BL21(DE3) strain (Stratagene). The cells were cultured at 37°C until OD600 reached 0.8, and then the protein expression was induced with 0.2 mM IPTG overnight at 18°C. The hexa-His-Sumo tagged protein was purified using a HisTrap FF column (GE Healthcare). The tag was cleaved by Ulp1 protease and further removed by a second-step HisTrap FF column (GE Healthcare) purification. The target protein was further purified by a Hiload Superdex G200 16/60 column (GE Healthcare).
7
Purification of AIPP3-BAH Recombinant Protein
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The sequence containing the AIPP3-BAH domain (residues 112–279) was constructed into a self-modified pMal-p2X vector to fuse a hexahistidine tag plus a maltose-binding protein (MBP) tag to the N-terminus of the target protein. The plasmids were transformed into Escherichia coli strain BL21 (DE3) RIL (Stratagene). Expression was induced at 16 °C overnight with 0.2 mM of IPTG. The recombinant proteins were purified with a prepackaged HisTrap FF column (GE Healthcare). The His-MBP tags were cleaved by TEV protease overnight and removed by flowing through a HisTrap FF column (GE Healthcare) again. The target protein was further purified using a Heparin column (GE Healthcare) and a Superdex G200 column (GE Healthcare). All the AIPP3-BAH mutations were generated by standard PCR-based mutagenesis procedure. The mutations of AIPP3-BAH and truncated AIPP2/PAIPP2 fragments were purified using the same protocols, as those used for the wild-type AIPP3-BAH. For the GST-AIPP3-BAH proteins used in histone peptide pull-down assays, the wild-type and mutated AIPP3-BAH proteins were purified with glutathione-Sepharose (GE Healthcare) and eluted with elution buffer (50 mM Tris-HCl pH 8.0, and 10 mM reduced glutathione). The peptides were purchased from GL Biochem or EpiCypher.
8
Purification of NudC Protein from E. coli
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The E. coli NudC gene was inserted into the pET-Sumo expression vector (Invitrogen) which fuses a hexa-His tag plus a yeast sumo tag at the N-terminus. The plasmid was transformed into E. coli BL21 (DE3) strain (Stratagene). The cells were cultured at 37 °C until OD600 reached 0.8, and then the protein expression was induced with 0.2 mM IPTG at 18 °C overnight. The hexa-His-Sumo tagged protein was purified using a HisTrap FF column (GE Healthcare). The tag was cleaved by Ulp1 protease and further removed by a second step HisTrap FF column (GE Healthcare) purification. The target protein was further purified by a Hiload Superdex G200 16/60 column (GE Healthcare). The purified protein was of high purity (above 95%) as shown by SDS-PAGE.
9
Purification of NudC Protein from E. coli
10
Purification and Characterization of AIPP3-BAH Domain
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The sequence containing the AIPP3-BAH domain (residues 112-279) was constructed into a self-modified pMal-p2X vector to fuse a hexahistidine tag plus a maltose-binding protein (MBP) tag to the N-terminus of the target protein. The plasmids were transformed into Escherichia coli strain BL21 (DE3) RIL (Stratagene). Expression was induced at 16 °C overnight with 0.2 mM of IPTG. The recombinant proteins were purified with a pre-packaged HisTrap FF column (GE Healthcare). The His-MBP tags were cleaved by TEV protease overnight and removed by flowing through a HisTrap FF column (GE Healthcare) again. The target protein was further purified using a Heparin column (GE Healthcare) and a Superdex G200 column (GE Healthcare). All the AIPP3-BAH mutations were generated by standard PCR-based mutagenesis procedure. The mutations of AIPP3-BAH and truncated AIPP2/PAIPP2 fragments were purified using the same protocols as those used for the wild-type AIPP3-BAH. For the GST-AIPP3-BAH proteins used in histone peptide pull-down assays, the wild-type and mutated AIPP3-BAH proteins were purified with glutathione-Sepharose (GE Healthcare) and eluted with elution buffer (50 mM Tris-HCl pH 8, and 10 mM reduced glutathione).
The peptides were purchased from GL Biochem or EpiCypher.
The peptides were purchased from GL Biochem or EpiCypher.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!