The human plasma was used to purify the ATIII by utilizing a 5 ml HiTrap Heparin HP affinity column (Cytiva). Human plasma was diluted with 1X PNE buffer (4.4 mM NaH2PO4.H2O, 15 mM Na2HPO4, 100 mM, 200 mM EDTA) in a 1:2 ratio and then loaded onto the Hi-Trap affinity chromatography column [20 (link)]. Before loading the plasma, the column was equilibrated with 1× PNE and then eluted with different NaCl concentration gradients (0–3.0 M). Eluted fractions were run on 10% SDS-PAGE to check the purity of the ATIII (Supplementary Figure S3). Human plasma was obtained from Rotary Bank, New Delhi, and ethical clearance for the same was obtained from the Institute ethical committee, JMI.
Hitrap heparin hp
Manufactured by Cytiva
Sourced in United States
The HiTrap Heparin HP is a pre-packed chromatography column designed for the high-performance purification of heparin-binding proteins. It utilizes a high-capacity heparin matrix to selectively capture and separate these target proteins from complex mixtures.
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5 protocols using hitrap heparin hp
1
Purification of Antithrombin III from Human Plasma
2
Purification of Recombinant RfxCas13d
3
Expression and Purification of CtNcs2/6 Complex
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The sequences of C. thermophilum (Ct) Ncs2 (UniProt ID: G0SFJ9) and CtNcs6 (UniProt ID: G0SAK0) were codon-optimized for expression in Escherichia coli, obtained from Genscript, and cloned into pETM30 (providing a TEV cleavable N-terminal 6xHis- and GST- tag) vectors. The proteins were expressed in BL21 (DE3) pRARE in LB at 18 °C using overnight induction with 0.5 M IPTG. Bacterial pellets were resuspended in lysis buffer (50 mM Tris–HCl pH 8, 300 mM NaCl, 0.15 % TX-100, 10 mM MgSO4, 1 mM DTT, 10 mg/ml DNase, 1 mg/ml lysozyme, 10 % glycerol and a protease inhibitor cocktail) and lysed to homogeneity using a high-pressure homogenizer Emulsiflex C3 (Avestin). To form the CtNcs2/6 complex, pellets containing each construct were mixed during lysis. The proteins were purified using GSTrap (Cytiva) columns on an ÄKTAstart (Cytiva) under standard conditions. Tags were optionally cleaved with TEV protease and removed in a second GSTrap purification step. Subsequently, the proteins were purified from nucleic acid contaminants using a HiTrap Heparin HP (Cytiva) column. Finally, proteins were purified by size-exclusion chromatography on a HiLoad 26/600 Superdex 200 prep-grade column (Cytiva). Purified proteins were stored at −80 °C in storage buffer (20 mM Tris pH 8, 150 mM NaCl and 1 mM DTT).
4
Chemokine Binding to Heparin Affinity
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Chemokine binding to a heparin matrix was performed on an ÄKTA Pure 25M2 System equipped with an external sample pump (GE Healthcare, Chicago, IL, USA) using a HiTrap Heparin HP (Cytiva Life Sciences, Glattbrugg, Switzerland) column. Lyophilized chemokines were reconstituted in sterile-filtered PBS (Fisher BioReagents, Reinach, Switzerland), and 50 µg of chemokine per run were automatically loaded onto the HiTrap Heparin HP column using a sample loop. After washing, column elution was performed with gradually increasing concentrations of PBS containing 2 M NaCl (Sigma). Chemokine binding strength was determined by comparing the conductivity during the peak elution at OD280nm. Analyzed peaks were normalized to their maximal absorption at 280 nm.
5
Purification of Schizosaccharomyces pombe Mag1
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Schizosaccharomyces pombe Mag1 (spMag1) was expressed and purified according to a reported method with modifications (43 (link)). Briefly, the bacterial expression plasmid spMag1-pBG100 was a kind gift from Dr Brandt Eichman. SpMag1 was overexpressed in BL21(DE3)-competent Escherichia coli cells (C2527H, NEB). Cells were grown at 25°C with shaking at 250 rpm until the optical density (OD) reached 1.0. Cultures were grown for 16 h at 16°C upon the addition of 0.5 mM isopropyl β-d -1-thiogalactopyranoside. Cells were lysed at 4°C in a lysis buffer (50 mM Tris–HCl, pH 8.0, 500 mM NaCl, 5% glycerol, 5 mM β-mercaptoethanol with protease inhibitor cocktail). SpMag1 was purified via a nickel-affinity column (HisTrap HP, 5 ml, Cytiva) and a heparin column (HiTrap Heparin HP, 1 ml, Cytiva). The pooled fractions were stored at −80°C.
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Schizosaccharomyces pombe Mag1 (spMag1) was expressed and purified according to a reported method with modifications (43 (link)). Briefly, the bacterial expression plasmid spMag1-pBG100 was a kind gift from Dr Brandt Eichman. SpMag1 was overexpressed in BL21(DE3)-competent Escherichia coli cells (C2527H, NEB). Cells were grown at 25°C with shaking at 250 rpm until the optical density (OD) reached 1.0. Cultures were grown for 16 h at 16°C upon the addition of 0.5 mM isopropyl β-
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