Hitrap nhs sepharose column

Manufactured by GE Healthcare

Sourced in Sweden

The HiTrap NHS-activated Sepharose column is a pre-packed, ready-to-use column designed for the covalent immobilization of ligands containing primary amino groups. The column matrix is based on highly cross-linked agarose beads, which provide a porous structure and high chemical and physical stability. The N-hydroxysuccinimide (NHS) ester groups on the column surface allow for the coupling of ligands to the matrix, enabling the capture and purification of target molecules from complex samples.

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Lab products found in correlation

Hitrap, by Cytiva (5 mentions) Kta system, by GE Healthcare (3 mentions) Escherichia coli bl21 star de3, by New England Biolabs (3 mentions) Acrodisc, by Cytiva (3 mentions) Freund s complete adjuvant, by Fujifilm (1 mentions) Freund s incomplete adjuvant, by Fujifilm (1 mentions) Complete edta free protease inhibitor cocktail, by Roche (1 mentions) Bl21 star de3, by Merck Group (1 mentions)

Spelling variants of this product

HiTrap columns (11 citations) NHS-Sepharose (8 citations) HiTrap NHS column (6 citations) NHS column (5 citations)

5 protocols using hitrap nhs sepharose column

Production and Purification of Affibody Constructs

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The affibody constructs were expressed at 37 °C in shake flask cultures of Escherichia coli BL21 Star (DE3) (New England Biolabs) in tryptic soy broth containing 100 µg/mL ampicillin. When OD₆₀₀ was between 0.6 and 1, protein expression was induced by addition of 1 mM isopropyl-β-D-1-thiogalactopyranoside (Appolo Scientific, Stockport, UK). Protein production was carried out for 3 h, after which the cells were harvested by centrifugation and lysed by sonication. The supernatants were clarified by centrifugation and filtration through a 0.45 µm Acrodisc syringe filter (Pall, Port Washington, NY, USA). The recombinantly expressed affibody constructs were purified by affinity chromatography on a HiTrap NHS sepharose column (GE Healthcare, Uppsala, Sweden) with immobilized human serum albumin (HSA) using an ÄKTA system (GE Healthcare), essentially as previously described [44 (link)] including elution with 500 mM acetic acid, pH 2.6. The fractions containing affibody constructs were pooled and lyophilized.
Z_HER2:V2 was produced and purified as previously described [45 (link)].

Xu T., Ding H., Vorobyeva A., Oroujeni M., Orlova A., Tolmachev V, & Gräslund T. (2020). Drug Conjugates Based on a Monovalent Affibody Targeting Vector Can Efficiently Eradicate HER2 Positive Human Tumors in an Experimental Mouse Model. Cancers, 13(1), 85.

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Antigen Immunization and IgG Purification

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The NetB toxin was used for antigen immunization as described elsewhere [2 (link)]. Briefly, the toxin (200 µg/ml) was detoxified by
treatment with formalin at a final concentration of 0.4% (v/v) and kept at 37°C for 7
days. After intraperitoneal administration of 20 µg of toxoid to mice (ddY strain, male 4
weeks old; SLC Co., Ltd., Hamamatsu, Japan), the animals were kept under observation for 4
days to examine their survival status. For the first immunization, rabbits (Japanese
white, male, 14 weeks old, Oriental Yeast, Tokyo, Japan) were injected with 20 µg of
toxoid intradermally, emulsified in an equal volume of Freund’s complete adjuvant (Wako
Pure Chemical Co., Osaka, Japan). Subsequently, the animals were injected with the same
dose of toxoid emulsified in an equal volume of Freund’s incomplete adjuvant (Wako Pure
Chemical Co.) intradermally 3 times every 2 weeks. Two weeks after the fourth
immunization, 20 µg of NetB toxoid alone was inoculated subcutaneously as a booster. Two
weeks after the booster, whole blood was collected from the heart under anesthesia, and
serum was collected. The IgG fraction was isolated from the rabbit serum as described by
Harlow and Lane [18 (link)]. Thereafter, the IgG against
the toxin was purified with a HiTrap NHS Sepharose column (GE Healthcare) according to the
manufacturer’s instructions.

ISLAM A.A., NAKATANI M., NAKAJIMA T., KOHDA T, & MUKAMOTO M. (2020). The cytotoxicity and molecular mechanisms of the Clostridium perfringens NetB toxin. The Journal of Veterinary Medical Science, 83(2), 187-194.

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Production and Purification of Affibody Fusion Proteins

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The affibody/ABD fusion proteins were expressed in Escherichia coli BL21 Star (DE3) (New England Biolabs), grown in tryptic soy broth supplemented with 5 g/L yeast extract and 100 μg/mL ampicillin. The cells were grown at 37 °C in shake flasks. Expression was induced by adding 1 mM of isopropyl β-d-1-thiogalactopyranoside (Appolo Scientific, Stockport, UK) when the OD₆₀₀ was between 0.6 and 1.0. The cells were cultured for another 3 h at 37 °C. The cell suspension was centrifuged and the cytoplasmic fraction was released by sonication. The cell lysate was clarified by passage through a 0.45 µm Acrodisc syringe filter (Pall, Port Washington, NY, USA). Human serum albumin-based affinity chromatography on a HiTrap NHS sepharose column (GE Healthcare, Uppsala, Sweden) was performed to isolate the ABD fused affibody constructs. The purification was carried out on an ÄKTA system (GE Healthcare Life Sciences, Uppsala, Sweden), essentially as previously described [20 (link)]. Elution was carried out with 500 mM acetic acid (pH = 2.6). The fractions containing affibody fusion proteins were pooled and lyophilized.

Ding H., Xu T., Zhang J., Tolmachev V., Oroujeni M., Orlova A., Gräslund T, & Vorobyeva A. (2021). Affibody-Derived Drug Conjugates Targeting HER2: Effect of Drug Load on Cytotoxicity and Biodistribution. Pharmaceutics, 13(3), 430.

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Protein Expression and Purification in E. coli

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Escherichia coli (BL21 Star (DE3)) (Merck Millipore, Billerica, Massachusetts, USA) was used as a host for protein expression. Overnight cultures of cells harboring the expression plasmids were prepared and diluted 1:100 in tryptic soy Broth (TSB) medium with yeast extract [31 (link)] and grown at 37 °C. When OD₆₀₀ reached approximately 1.5, protein expression was induced by addition of Isopropyl β-D-1-thiogalactopyranoside to a final concentration of 1 mM. The cells were grown a further 2.5 h, after which they were harvested by centrifugation. The cell pellets were subsequently resuspended in TST-buffer (25 mM Tris(hydroxymethyl) aminomethane, 1 mM EDTA, 200 mM NaCl, 0.05% Tween 20 pH 8.0) supplemented with Complete EDTA-free protease inhibitor cocktail (Roche Diagnostic, Basel, Switzerland) and then broken by sonication. The fusion toxins were purified on an HiTrap NHS sepharose column (GE Healthcare Bio-Sciences, Uppsala, Sweden) with immobilized HSA, as previously described [31 (link)]. The proteins were eluted with 0.5 M acetic acid (pH 2.6) followed by buffer exchange to phosphate buffered saline (PBS). The molecular masses of the eluted proteins were determined by liquid chromatography electrospray ionization mass spectrometry on a Bruker impact II instrument (Agilent Technologies, Santa Clara, CA, USA), as previously described [31 (link)].

Ding H., Altai M., Yin W., Lindbo S., Liu H., Garousi J., Xu T., Orlova A., Tolmachev V., Hober S, & Gräslund T. (2020). HER2-Specific Pseudomonas Exotoxin A PE25 Based Fusions: Influence of Targeting Domain on Target Binding, Toxicity, and In Vivo Biodistribution. Pharmaceutics, 12(4), 391.

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Affibody Production in E. coli

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The affibody constructs were expressed at 37 °C in shake flask cultures of Escherichia coli BL21 Star (DE3) (New England Biolabs). When OD₆₀₀ was between 0.6 and 1, protein expression was induced by addition of 1 mM isopropyl β-D-1-thiogalactopyranoside (Appolo Scientific, Stockport, UK). Protein production was carried out for 3 h, after which the cells were harvested by centrifugation and lysed by sonication. The supernatants were clarified by centrifugation and filtration through a 0.45 μm Acrodisc syringe filter (Pall, Port Washington, NY, USA). The recombinantly expressed affibody constructs were purified by affinity chromatography on a HiTrap NHS sepharose column (GE Healthcare, Uppsala, Sweden) with immobilized human serum albumin (HSA) using an ÄKTA system (GE Healthcare), essentially as previously described [14 (link)] including elution with 50 mM acetic acid. The fractions containing affibody constructs were pooled and lyophilized.

Ding H., Altai M., Rinne S.S., Vorobyeva A., Tolmachev V., Gräslund T, & Orlova A. (2019). Incorporation of a Hydrophilic Spacer Reduces Hepatic Uptake of HER2-Targeting Affibody–DM1 Drug Conjugates. Cancers, 11(8), 1168.

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