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Hitrap blue hp column

Manufactured by GE Healthcare
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Sourced in Japan

The HiTrap Blue HP column is a pre-packed affinity chromatography column designed for the purification of a variety of proteins, including enzymes, hormones, and other biomolecules. The column contains a ligand that selectively binds to target proteins, allowing for their separation and purification from complex mixtures. The column is suitable for use in both small-scale and large-scale applications.

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Lab products found in correlation

Akta prime, by GE Healthcare (1 mentions) Hitrap q hp column, by GE Healthcare (1 mentions) Finasteride, by Merck Group (1 mentions) Cortisone, by Steraloids (1 mentions) Histrap fast flow column, by GE Healthcare (1 mentions) Ni nta agarose resin, by Qiagen (1 mentions) Chelating sepharose fast flow resin, by GE Healthcare (1 mentions) General chemicals, by Merck Group (1 mentions) Restriction enzyme, by New England Biolabs (1 mentions)

5 protocols using hitrap blue hp column

1

Affinity Chromatography of ASP0-50%

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ASP0-50% was dissolved in approximately 1 ml of 20 mM HEPES buffer (pH 7.4), and dialyzed against 20 mM HEPES buffer (pH 7.4). After dialysis, sample (total protein amount of 42 mg) was applied to a HiTrap Blue HP column (5 ml, GE Healthcare, Tokyo, Japan) equilibrated with 20 mM HEPES buffer (pH7.4) using a manual syringe. After a 50-ml wash with 20 mM HEPES buffer (pH 7.4), the elution of proteins bound to the column was performed with a step-wise elution with 20 mM HEPES buffer (pH 7.4) containing 2 M NaCl, and 1 ml of fractions were collected. Aliquot of fractions from the column unbound (Blue pass) and bound elutes (Blue elute) were subjected to the AOND assay and protein content analysis (S1 Fig).
Kanno T., Yasutake K., Tanaka K., Hadano S, & Ikeda J.E. (2017). A novel function of N-linked glycoproteins, alpha-2-HS-glycoprotein and hemopexin: Implications for small molecule compound-mediated neuroprotection. PLoS ONE, 12(10), e0186227.
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2

Wnt3a Purification from Conditioned Media

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Processed CM was applied to a 5 ml bed volume HiTrap Blue HP column (GE Healthcare). The column was protected from residual precipitate by an inline 0.45 μm syringe filter. The sample was applied to the column at ~2 ml/min with a peristaltic pump. The column eluate was collected during loading and α-Wnt3a immunoblot analysis performed to monitor Wnt3a binding to the column matrix. After loading up to 5 L processed CM and washing with 40 ml 20 mM Tris HCl, pH 7.45 containing 0.14 M NaCl and 1 % (w/v) CHAPS detergent (buffer A) the column was placed in an AKTA Prime FPLC system (GE Healthcare). All operations to this point were carried out at 4 °C. The column was washed with 150 ml buffer A at 2.5 ml/min and protein eluted with 1.5 M sodium chloride, 20 mM Tris-HCl, 1% CHAPS, pH 7.45 (buffer B) using the following gradient: 20 ml to 25% B; 5 ml to 35% B; 2.5 ml to 100% B followed by a further 75 ml at 100% B (flow rate = 2.5 ml/min). The collected fractions were pooled based on A280 and SDS-PAGE analysis.
Witkowski A., Krishnamoorthy A., Su B., Beckstead J.A, & Ryan R.O. (2014). Isolation and characterization of recombinant murine Wnt3a. Protein expression and purification, 0, 41-48.
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3

Purification of Recombinant Erythropoietin

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The purified pcDNA 3.3 expression vector containing a sequence encoding EPO-WT or EPO mutant was transiently transfected into FreeStyle 293-F cell using 25 kD linear polyethylenimine (Polysciences). After 6 days, EPO protein was purified from culture supernatant using Hitrap Blue HP column (GE Healthcare), eluted with 1.5 M NaCl, and buffer exchanged into 20 mM Tris-HCI pH 8.45. Next, sample was loaded onto Hitrap Q HP column (GE Healthcare). The column was washed with 20 mM sodium acetate pH 4.00 followed by second wash with 20 mM Tris. EPO protein was then eluted with 1 M NaCl. Purified EPO protein was buffer exchanged into a storage buffer (50 mM sodium phosphate buffer, pH 7.0, 1.5% glycine and 0.003% tween-20).
Susantad T., Fuangthong M., Tharakaraman K., Tit-oon P., Ruchirawat M, & Sasisekharan R. (2021). Modified recombinant human erythropoietin with potentially reduced immunogenicity. Scientific Reports, 11, 1491.
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4

Cortisone and 7α-Hydroxycholest-4-en-3-one Synthesis

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Cortisone and 7α-hydroxycholest-4-en-3-one were purchased from Steraloids. Finasteride was purchased from Sigma-Aldrich. NADPH was purchased from EMD. NADP+ was purchased from Roche Applied Science. E. coli strain C41 (DE3) was provided by Dr. J. E. Walker (Medical Research Council Laboratory of Molecular Biology, Cambridge, UK). HisTrap Fast Flow column (5 mL) and HiTrap Blue HP column (5 mL) were purchased from GE Healthcare. All other reagents were of American Chemical Society quality or higher.
Chen M., Jin Y, & Penning T.M. (2015). In-Depth Dissection of the P133R Mutation in Steroid 5β-Reductase (AKR1D1): A Molecular Basis of Bile Acid Deficiency. Biochemistry, 54(41), 6343-6351.
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5

Protein Purification and Analysis

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General chemicals and the phosphate assay kit (MAK308) were from Sigma-Aldrich. Restriction enzymes and commercial apyrase were from New England Biolabs. Chelating Sepharose fast flow resin and the pre-packed HiTrapBlue HP column were from GE Healthcare or Cytiva. Ni-NTA agarose resin was from Qiagen. Coelenterazine H BRET substrate was from Nanolight. Dialysis membrane was from Spectrum Labs.
Karim J.A., Lambert N.A, & Pioszak A.A. (2022). Time- and cost-efficient bacterial expression and purification of potato apyrase. Protein expression and purification, 203, 106215.
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