Cibacron blue 3ga agarose

Manufactured by Merck Group

Sourced in United States

Cibacron Blue 3GA agarose is a type of affinity chromatography resin. It is used for the purification and isolation of a wide range of biomolecules, including enzymes, proteins, and other macromolecules. The Cibacron Blue dye is covalently attached to the agarose beads, providing a specific binding site for the targeted biomolecules.

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3 protocols using cibacron blue 3ga agarose

Purification and Quantification of LPO

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LPO from bovine milk (ε412 = 112,000 M⁻¹ cm⁻¹ (51 )), Hank’s balanced salt solution and PBS for cell culture, Cibacron Blue 3GA agarose (type 3000-CL), 1-chloro-2,4-dinitrobenzene, sodium thiocyanate, NADH, EDTA, IAM, EC-oxyrase, calcium chloride dihydrate, spectinomycin dihydrochloride pentahydrate, Vivaspin 500 (MWCO = 3 kDa), and Amicon Ultra 0.5 centrifugal filter units (MWCO = 10 kDa) were purchased from Sigma–Aldrich (Merck). Competence stimulating peptide-1 was purchased from AnaSpec. Bovine serum albumin was from Gibco (Thermo Fisher Scientific). Auranofin was from Santa Cruz Biotechnology. H₂O₂ (30%) (ε₂₄₀ = 43.6 M⁻¹ cm⁻¹ (52 (link))) was from LabServ. 2-Nitro-5-thiobenzoate (TNB) was prepared from 5,5′-dithiobis-(2-nitrobenzoic acid) (Sigma–Aldrich) through alkaline hydrolysis as described (53 (link)). NADPH was purchased from Carbosynth and trypsin (sequencing grade) from Promega. HOSCN was generated and quantified as described (21 (link)), kept on ice, and used within 30 min of quantification.

Shearer H.L., Pace P.E., Paton J.C., Hampton M.B, & Dickerhof N. (2022). A newly identified flavoprotein disulfide reductase Har protects Streptococcus pneumoniae against hypothiocyanous acid. The Journal of Biological Chemistry, 298(9), 102359.

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Purification of GroEL Chaperone Protein

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Lyophilized GroEL (Cat # C7688, Sigma-Aldrich, St. Louis, MO, USA) was solubilized in buffer containing 50 mM TRIS pH 7.5, 10 mM KCl and 10 mM MgCl₂. The solubilized protein was subjected to size-exclusion chromatography on a Superdex 200 10/30 column connected to a ÅKTA FPLC (GE, Pittsburgh, PA, USA). The elution volume fractions corresponding to the appropriate molecular weight of GroEL were pooled and concentrated using an Amicon (EMD Millipore, Billerica, MA, USA) concentrator with a 100,000 Da cut-off. The concentrated protein was thereafter added to an equal volume of pre-swelled Cibacron Blue 3G-A agarose (Cat # B1064, Sigma-Aldrich, St. Louis, MO, USA) in batch-mode at 4 °C overnight. Centrifuging the suspension at 20,000× g and collecting the supernatant recovered the GroEL. Protein concentration was measured as 2.5 mg/mL by monitoring the O.D.₂₈₀ absorbance.
Homomeric rat GluK2 and GluA2 were expressed, purified and functionally trapped as described in Meyerson et. al. 201419 (link).

Meyerson J.R., Rao P., Kumar J., Chittori S., Banerjee S., Pierson J., Mayer M.L, & Subramaniam S. (2014). Self-assembled monolayers improve protein distribution on holey carbon cryo-EM supports. Scientific Reports, 4, 7084.

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Purification of Human IgA1 Antibody

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Serum albumin was removed by pretreatment with Cibacron Blue 3GA Agarose (Sigma, St. Louis, MO). A prepacked Cibacron Blue column (1 mL) was washed with a mixture of 1 M NaCl and 0.1 M borate (10 mL, pH 9.8) followed by 10 mL distilled water, and then equilibrated with 10 mM Tris-HCl (10 mL, pH 7.4). The flow-through fraction and wash were collected and combined as the IgA1 fraction, and then purified by affinity chromatography using an anti-human IgA1 monoclonal antibody (anti-IgA1 mAb 7303B, Institute of Immunology, Tokyo, Japan) coupled to cyanogen bromide-activated Sepharose 4FF (1 mg IgG/mL gel; Buckinghamshire, UK). The fraction was loaded onto the anti-IgA1 mAb-coupled Sepharose 4B column (2 mL; GE Healthcare) and washed with 20 mL PBS. The bound IgA1 was eluted with 0.1 M glycine (pH 2.5) and immediately neutralized with 1 M Tris-HCl (pH 8.0). The remaining IgG was removed by mixing the eluate with a protein G bead suspension (Thermo Fisher Scientific, Waltham, MA) and incubating at 4°C overnight. The supernatant was collected, dialyzed against 10 mM ammonium bicarbonate, and then lyophilized.

Narimatsu Y., Kuno A., Ito H., Kaji H., Kaneko S., Usui J., Yamagata K, & Narimatsu H. (2014). IgA Nephropathy Caused by Unusual Polymerization of IgA1 with Aberrant N-Glycosylation in a Patient with Monoclonal Immunoglobulin Deposition Disease. PLoS ONE, 9(3), e91079.

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