Histrap

Manufactured by GE Healthcare

Sourced in United States, Germany, United Kingdom

HisTrap is a column product designed for the purification of histidine-tagged proteins. It utilizes Ni Sepharose resin to bind and capture target proteins expressing a histidine tag. The column can be used in standard liquid chromatography systems to efficiently isolate and concentrate desired proteins from complex mixtures.

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Lab products found in correlation

114 protocols using histrap

Purification and Labeling of FUS and hnRNPA2

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FLAG-tagged DPR₅₀ expression constructs were synthesized by Genscript (Piscataway, USA). The DPR₁₀₀ constructs were a kind gift of Dr. Daisuke Ito (Department of Neurology, Keio University, Tokyo, Japan).
Peptides were chemically synthesized by Pepscan (Lelystad, Netherlands). Peptides were dissolved in milli-Q water and stored at −80°C. Peptides were fluorescently labeled using Alexa Fluor Labeling Kits (Thermo Scientific).
FUS LC domain was expressed in E. coli and purified as described previously (Burke et al., 2015 (link)). hnRNPA2 LC domain (residues 190-341) was expressed with a TEV-cleavable N-terminal hexahistidine tag, purified from the inclusion body via HisTrap (GE Healthcare) in urea containing buffers, concentrated, diluted into native buffer for TEV cleavage of hexahistidine tag, resolublized by addition of solid urea, separated from his-tagged TEV protease and cleaved his-tag by HisTrap, and concentrated to 1-2 mM in 8M urea 20mM MES pH 5.5. hnRNPA2 was aliquoted and flash frozen. Experiments were conducted by dilution into native buffer conditions.

Boeynaems S., Bogaert E., Kovacs D., Konijnenberg A., Timmerman E., Volkov A., Guharoy M., De Decker M., Jaspers T., Ryan V.H., Janke A.M., Baatsen P., Vercruysse T., Kolaitis R.M., Daelemans D., Taylor J.P., Kedersha N., Anderson P., Impens F., Sobott F., Schymkowitz J., Rousseau F., Fawzi N.L., Robberecht W., Van Damme P., Tompa P, & Van Den Bosch L. (2017). Phase Separation of C9orf72 Dipeptide Repeats Perturbs Stress Granule Dynamics. Molecular Cell, 65(6), 1044-1055.e5.

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Recombinant Expression and Purification of NoV P-Domain

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Each of GII.3 (TCH04-577) and GII.4 Sydney 2012 P-domain sequence was cloned into the expression vector pMal-C2E (New England BioLabs). The recombinant P-domain was expressed with an N-terminal His6-maltose-binding protein (MBP) tag, and a tobacco etch virus (TEV) cleavage site between the MBP and P-domain in E. coli BL21(DE3) and purified using His-Trap (GE Healthcare). The His-MBP tag was then removed using TEV protease and separated from the P-domain by purifying it once again using His-Trap (GE Healthcare), MBPTrap (GE Healthcare) affinity columns, and size exclusion chromatography as previously described^{61 (link)}. The purified P-domain was concentrated and stored in a buffer containing 20 mM Tris-HCl (pH 7.2), 150 mM NaCl, and 2.5 mM MgCl₂. The recombinant M4 was expressed in E. coli WK6 strain. The periplasmic proteins were extracted by osmotic shock using Tris/EDTA/Sucrose (TES) buffer, and His-tagged M4 was purified from the periplasmic extract using a High-Trap HP Ni-chelating column (GE Healthcare, US).

Salmen W., Hu L., Bok M., Chaimongkol N., Ettayebi K., Sosnovtsev S.V., Soni K., Ayyar B.V., Shanker S., Neill F.H., Sankaran B., Atmar R.L., Estes M.K., Green K.Y., Parreño V, & Prasad B.V. (2023). A single nanobody neutralizes multiple epochally evolving human noroviruses by modulating capsid plasticity. Nature Communications, 14, 6516.

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Nanobody-Tub-tag Protein Expression

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Example 7

Nanobody-Tub-tag fusion proteins were expressed in E. coli (JM109). Cells were induced with 0.5 mM IPTG and incubated at 18° C. for 18 h. Lysis was performed in presence of Lysozyme (100 μg/ml), DNAse (25 μg/ml) and PMSF (2 mM) followed by sonification (Branson® Sonifier; 16×8 sec, 20% Amplitude) and debris centrifugation at 20.000 g for 30 min. The protein was purified with an Äkta FPLC system using a 5 ml His-Trap (GE Healthcare) column, peak fractions were concentrated to 2 ml using Amicon filter columns (Cut-off 3 kDa; (Millipore)) and subjected to size exclusion chromatography using a Superdex 75 column (GE Healthcare). Peak fractions were pooled and protein aliquots were shock-frozen and stored at −80° C. at 0.5 g/l. Note: Tub-tag is shown in SEQ ID No. 3.

US10208084B2. Means and methods for site-specific functionalization of polypeptides (2019-02-19). LUDWIG-MAXIMILIANS-UNIVERSITÄT MÜNCHEN [DE], FORSCHUNGSVERBUND BERLIN E.V. [DE]. Inventors: Heinrich Leonhardt [DE], Jonas Helma [DE], Dominik Schumacher [DE], Christian Hackenberger [DE].

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Determination of Ligand-Kinase Crystal Structures

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Experiments involving target-drug complex formation, data collection, and data processing were done using the standard operating protocol as validated previously^{6 (link)} for the determination of atomic resolution ligand–kinase crystal structures. Protein purification^{6 (link)} was done using a combination of affinity-based adsorption chromatography (HisTrap™; GE Healthcare), gel filtration (HiPrep 26/10 column; GE Healthcare) and ion exchange chromatography (Q Sepharose™; GE Healthcare).

Roy S.M., Grum-Tokars V.L., Schavocky J.P., Saeed F., Staniszewski A., Teich A.F., Arancio O., Bachstetter A.D., Webster S.J., Van Eldik L.J., Minasov G., Anderson W.F., Pelletier J.C, & Watterson D.M. (2015). Targeting Human Central Nervous System Protein Kinases: An Isoform Selective p38αMAPK Inhibitor That Attenuates Disease Progression in Alzheimer’s Disease Mouse Models. ACS Chemical Neuroscience, 6(4), 666-680.

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Crystallization of CD27 and mAb 2177 Fab

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The CD27 construct used for crystallization contained amino acids 1–101 of human CD27 (21–121 of the full-length sequence according to the UniProtKB entry CD27_HUMAN) with a 6×His tag at the C-terminus. The protein was expressed in baculovirus-infected Sf9 insect cells (Spodoptera frugiperda) and was purified at Proteos Inc. (Kalamazoo, Michigan, USA) using metal-ion chromatography on an Ni–NTA column (Thermo Fisher) and size-exclusion chromatography (SEC) on a Superdex 200 column (GE Healthcare). It was further purified in-house on a Mono S column (GE Healthcare). No deglycosylation was attempted.
The Fab fragment of mAb 2177 was constructed by fusing the mouse variable domains with human IgG1/κ constant domains that contained a 6×His tag at the C-terminus of the heavy chain. Two Lonza-based vectors (Lonza Group, Switzerland), p4275 and p4208, were used to construct expression plasmids for IgG1 heavy chain and κ light chain, respectively, following the protocol described previously (Zhao et al., 2009 ▸ ). The Fab was expressed in HEK 293 cells and was purified by affinity and size-exclusion chromatography using HisTrap and Superdex 200 columns, respectively (GE Healthcare).

Teplyakov A., Obmolova G., Malia T.J, & Gilliland G.L. (2017). Crystal structure of CD27 in complex with a neutralizing noncompeting antibody. Acta Crystallographica. Section F, Structural Biology Communications, 73(Pt 5), 294-299.

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Purification of D8 Protein from E. coli

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BL21-CodonPlus(DE3)-RIL competent cells (Agilent) were transformed with one of the D8 expression vectors and grown in LB media with 1 mM Ampicillin at 37°C until OD₆₀₀ ∼0.6. Protein expression was then induced with 1 mM IPTG for 4 hrs at 37°C, while shaking at 230 rpm. Cells were pelleted and resuspended in lysis buffer containing 100 mM Tris pH 8.0, 300 mM NaCl, 0.5 mM EDTA, 20 mM Imidazole, 0.2 mM PMSF and lysed under 20000 psi pressure using a microfluidizer (Microfluidics). Cell lysate was clarified at 50,000 g for 20 min. Supernatant was loaded onto 5 mL Ni-NTA column (His-Trap, GE). Bound D8 protein was eluted with 20 mM Tris pH 8.0, 300 mM NaCl, 200 mM Imidazole. After overnight dialysis against 20 mM Tris pH 8.0, 200 mM NaCl, the sample was concentrated and subjected to SEC using a Superdex 200 10/300GL column (GE) in the same buffer. The monomeric peak with V_E≅16.5 mL and oligomeric peak with V_E≅11.7 mL were collected in separate fractions.

Matho M.H., de Val N., Miller G.M., Brown J., Schlossman A., Meng X., Crotty S., Peters B., Xiang Y., Hsieh-Wilson L.C., Ward A.B, & Zajonc D.M. (2014). Murine Anti-vaccinia Virus D8 Antibodies Target Different Epitopes and Differ in Their Ability to Block D8 Binding to CS-E. PLoS Pathogens, 10(12), e1004495.

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B2.1A scFv Expression and Purification

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The genes encoding the variable heavy (vH) and variable light (vL) chain were generated by reverse transcription-polymerase chain reaction from total RNA of the B2.1A-secreting hybridomas cells. The vH and vL segments were linked via a nucleotide sequence encoding a flexible linker (GGSGGSGGGGSGGGGSGGGAS) and this cassette placed downstream from a Igk-leader sequence in a modified mammalian expression vector pDisplay (Life Technologies) to generate a single-chain variable fragment (scFv) expression construct. The vH-linker-vL cassette was subsequently sub-cloned into pLOU3, an Escherichia coli expression vector derived from the pMAL-c2x vector (NEB). The construct encodes a maltose-binding protein (MBP)-B2.1 A scFv fusion protein, which carries a 6-histidine sequence at its N-terminus and a tobacco etch virus (TEV) protease cleavage site at the C-terminus of MBP. Briefly, the fusion protein was expressed in E. coli strain Rosetta-gami 2(DE3)pLysS (EMD Millipore) and the protein purified from cell extract using HisTrap and MBPTrap columns (GE Healthcare). The protein was then digested with TEV protease and re-applied to the HisTrap column. The unbound fraction containing B2.1 A scFv was further purified on a 16/60 Sephacryl S100 (GE Healthcare) in PBS.

Cowton V.M., Owsianka A.M., Fadda V., Ortega-Prieto A.M., Cole S.J., Potter J.A., Skelton J.K., Jeffrey N., Di Lorenzo C., Dorner M., Taylor G.L, & Patel A.H. (2021). Development of a structural epitope mimic: an idiotypic approach to HCV vaccine design. NPJ Vaccines, 6, 7.

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Antibody Purification and Detection

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Antibodies against YAP and HA-F7 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti–FLAG-M2 and β-actin antibodies were from Sigma Aldrich. TAZ antibody was from BD Transduction Laboratories. Anti-His-HRP antibody was obtained from Miltenyi Biotec Inc. Restriction enzymes for cloning were purchased from NEB (New England Bio lab). HisTrap^™ and GSTrap^™ Fast Flow columns were from GE Healthcare. Protein G/A-Agarose was purchased from Roche Diagonistics.

Khanal P., Jia Z, & Yang X. (2018). Cysteine residues are essential for dimerization of Hippo pathway components YAP2L and TAZ. Scientific Reports, 8, 3485.

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Purification of HIV-1 IN-LEDGF/p75 Complex

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ALLINI BI/D, LEDGIN-6, ALLINI-2, and KF116 were synthesized as previously described.^{25 (link),29 (link),31 (link),40 (link)} WT HIV-1 IN and LEDGF/p75 recombinant proteins with 6 × His or FLAG tags were expressed in E. coli cells and purified as described.^{31 (link)} Expression and purification of the complex between His-LEDGF/p75 and FLAG-HIV-1 IN was performed as follows. His-LEDGF/p75 was expressed in E. coli from pFT-1-LEDGF derived from pRSETB (Invitrogen).^{31 (link)} FLAG-HIV-1 IN was expressed in E. coli as previously reported.^{31 (link)} Cell pellets of both were mixed and lysed by sonication in buffer containing 500 mM NaCl, 50 mM HEPES at pH 7.5, 2 mM β-mercaptoethanol, 20 mM Imidazole, and 1 × Complete protease inhibitor (Roche). After centrifugation, the soluble lysate was filtered and purified using a 5 mL HisTrap (GE Healthcare) chromatography column. Bound protein was eluted using a linear gradient of 20 mM to 500 mM imidazole in the same buffer. Fractions containing both proteins were pooled and concentrated using a 100 kDa MWCO spin column. The proteins were then subjected to size exclusion chromatography using a HiLoad 16/60 Superdex 200 (GE Heathcare) in 500 mM NaCl, 50 mM HEPES at pH 7.5, and 2 mM β-mercaptoethanol. Fractions that contained the complex between His-LEDGF/p75 and FLAG-HIV-1 IN were pooled and concentrated using a 100 kDa MWCO spin column.

Feng L., Dharmarajan V., Serrao E., Hoyte A., Larue R.C., Slaughter A., Sharma A., Plumb M.R., Kessl J.J., Fuchs J.R., Bushman F.D., Engelman A.N., Griffin P.R, & Kvaratskhelia M. (2016). The Competitive Interplay between Allosteric HIV-1 Integrase Inhibitor BI/D and LEDGF/p75 during the Early Stage of HIV-1 Replication Adversely Affects Inhibitor Potency. ACS chemical biology, 11(5), 1313-1321.

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Crystallization of Recombinant E. coli L-Asparaginase

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Recombinant Escherichia coli type II l-asparaginase (EcAII) was produced in recombinant form as described in Maggi et al.^{26 (link)}. Briefly, protein overexpression was obtained by the autoinducing method using E. coli BL21(DE3) ΔansA/ΔansB as a host strain. Protein purification was obtained by immobilized metal affinity (HisTrap, GE Healthcare) and anionic exchange chromatography (HiTrap Q, GE healthcare). Protein to be crystallized was buffer exchanged on column (HiTrap Desalting, GE Healthcare) against 50 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), pH 7.4. Protein solution pure to homogeneity was concentrated to 2.5 mg/ml and used for crystallization. Multiple prismatic crystals were obtained in condition n° 19 of Molecular Dimensions structure screening 2 (MD-02). The condition was further optimized and modified as follows: 0.1 M Sodium HEPES pH 8.5, 10% w/v PEG 8000, 5% v/v Ethylene glycol. Single prismatic crystals were obtained in a sitting drop setting at 21 °C after 4–5 days and mounted on nylon loops in a cryoprotectant solution containing the crystallization condition added with 25% v/v glycerol.

Maggi M., Meli M., Colombo G, & Scotti C. (2021). Revealing Escherichia coli type II l-asparaginase active site flexible loop in its open, ligand-free conformation. Scientific Reports, 11, 18885.

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