Hitrap cm ff

Manufactured by GE Healthcare

Sourced in Japan, United States

HiTrap CM FF is a pre-packed, ready-to-use cation exchange chromatography column designed for rapid and convenient purification of proteins and other biomolecules. The column utilizes a strong cation exchange resin, carboxymethyl (CM) Sepharose, for high-resolution separation of molecules based on their charge.

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Lab products found in correlation

Hitrap, by Cytiva (4 mentions) Hitrap sp hp, by GE Healthcare (1 mentions) Hitrap q ff column, by GE Healthcare (1 mentions) Akta prime plus system, by GE Healthcare (1 mentions) Akta prime plus, by Cytiva (1 mentions)

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HiTrap CM FF column (8 citations)

4 protocols using hitrap cm ff

Purification of Bacteriophage T4 Lysozyme

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The pellet of cells (3 g) that expressed the pET-gE plasmid was resuspended in 25 mL of buffer E (0.1 M sodium phosphate buffer, pH 6.6, 0.2 M NaCl, 10 mM MgCl₂ and 1 mM CaCl₂). Then, 0.5 mL of chloroform was added to this mixture and the suspension was stirred for 30 min. To reduce the viscosity, 1 mL of 1 M MgCl₂ and a few grains of DNase I were added, and the mixture was stirred for an additional 1.5 h. The lysates were centrifuged for 30 min at 28,000× g (the R18A rotor) to remove cell debris. The supernatants were dialyzed into buffer F (50 mM Tris pH7.25, 1 mM EDTA). The dialyzed sample was applied to the HiTrap CM FF (5 mL, GE healthcare) cation exchange column, pre-equilibrated with buffer F. The bound material was eluted by 50–300 mM NaCl linear gradient with the same buffer. Fractions containing T4L were pooled, dialyzed against 50 mM sodium phosphate buffer (pH 5.8), and then run through a HiTrap SP HP (1 mL, GE healthcare) cation exchange column equilibrated with the same buffer. T4L was eluted with buffer G (0.1 M sodium phosphate, pH 6.6, 0.55 M NaCl).

Kanamaru S., Uchida K., Nemoto M., Fraser A., Arisaka F, & Leiman P.G. (2020). Structure and Function of the T4 Spackle Protein Gp61.3. Viruses, 12(10), 1070.

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Purification of Alpha-1-Acid Glycoprotein

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AGP was purified from human plasma fraction V that provided by KAKETSUKEN, Kumamoto, Japan. Fraction V was dissolved in acetate buffer (10 mM) and applied to a HiTrap CM FF column (5 mL) and a HiTrap Q FF column (5 mL) on an AKTAprime Plus System (GE Healthcare, Tokyo, Japan). Bound material was subsequently eluted with acetate buffer (10 mM) containing NaCl (0.5 mol/L) at a flow rate of 5 mL/min. The eluate was dialyzed against deionized water at 4 °C, freeze-dried and stored at −20 °C. The purified protein was confirmed as AGP by Western blotting.

Bi J., Watanabe H., Fujimura R., Nishida K., Nakamura R., Oshiro S., Imafuku T., Komori H., Miyahisa M., Tanaka M., Matsushita K, & Maruyama T. (2018). A downstream molecule of 1,25-dihydroxyvitamin D3, alpha-1-acid glycoprotein, protects against mouse model of renal fibrosis. Scientific Reports, 8, 17329.

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Synthesis of T84.66-Gelonin Conjugate

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T84.66-gelonin conjugate was synthesized via a disulfide bond utilizing a PEG (NHS-PEG-OPSS, 2 kDa) cross-linker. T84.66 was conjugated with the PEG by mixing T84.66 (10 mg/mL in 20 mM PBS, pH 7) with 5-fold molar excess of NHS-PEG-OPSS and incubation for 3 h at RT. The unreacted PEG was removed by ultrafiltration (MWCO: 10 kDa), and the OPSS groups were quantified by P2T assay. To introduce thiol groups to gelonin, gelonin (5 mg/mL in 20 mM PBS buffer, 50 mM triethanolamine, 5 mM EDTA, pH 8.5) was mixed with 5-fold molar excess of Traut’s reagent (2-Iminothiolane) and incubated for 1 h at RT. After incubation, unreacted excessive Traut’s reagent was removed by ultrafiltration (MWCO: 10 kDa), and the thiol groups generated on gelonin were quantified by Ellman’s assay. The prepared T84.66-PEG-OPSS and thiol-activated gelonin (gelonin-SH) were mixed together by a 1:5 molar ratio and incubated for 4 h at 4°C. After incubation, the T84.66-gelonin was purified from unreacted T84.66-PEG-OPSS by using a cation exchange column (HiTrap CM-FF, GE Healthcare Bio-Sciences, Pittsburgh, PA) with elution by a salt gradient (0 to 2 M NaCl at a rate of 0.02 M/min, flow rate: 1 mL/min, detection: 280 nm). Any unreacted gelonin-SH contained in the T84.66-gelonin peak fraction was further removed from T84.66-gelonin by ultrafiltration (MWCO: 100 kDa).

Shin M.C., Zhang J., Ah Min K., Lee K., Moon C., Balthasar J.P, & Yang V.C. (2014). Combination of Antibody Targeting and PTD-Mediated Intracellular Toxin Delivery for Colorectal Cancer Therapy. Journal of controlled release : official journal of the Controlled Release Society, 194, 197-210.

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Reconstitution of Cardiac Troponin Complex

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Human cardiac troponin I (hcTnI (P19429, with all Cys residues mutated to Ala, named Cys-less TnI)) and human cardiac troponin T (hcTnT (P453796)) were kindly donated by Dr. Piotr Fajer (Institute of Molecular Biophysics, Florida State University). Proteins were expressed in BL21 E. coli. Recombinant TnI was purified using a weak cation exchange CM sepharose column (HiTrap CM FF, GE Healthcare, Fairfield, Connecticut, USA). Purification or recombinant TnT with an N-terminal His-tag began with a Ni column (Ni charged nitrilotriacetic acid, Amersham). The second purification step used a DE-52 anion exchange column after cleavage of the His-tag. The ternary troponin complex was reconstituted by combining the desired TnC construct with TnT and TnI at a molar ratio of 1:1:1.5. The proteins were mixed and dialyzed overnight against Reconstitution Buffer A (50mM MOPS, 0.5M KCl 1mM EDTA, 2mM CaCl₂, 3mM MgCl₂, 6M urea, pH = 7.2), followed by Reconstitution Buffer B (50mM MOPS, 0.5 M KCl, 2mM CaCl₂, 3mM MgCl₂, pH = 7.2). Finally, a two-step dialysis of 6hrs each against Reconstitution Buffer C (50mM MOPS, 0.1 M KCl, pH = 7.2) was performed. Reconstitution of the complex was evaluated by size exclusion chromatography (SEC; Superdex 200, 10/300 Cat. No. 17-5175-01, GE Healthcare Life Sciences).

Badr M.A., Pinto J.R., Davidson M.W, & Chase P.B. (2016). Fluorescent Protein-Based Ca2+ Sensor Reveals Global, Divalent Cation-Dependent Conformational Changes in Cardiac Troponin C. PLoS ONE, 11(10), e0164222.

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