Protein a sepharose column

Manufactured by Cytiva

Sourced in Sweden

The Protein-A Sepharose column is a chromatography product designed for the purification of antibodies. The column matrix consists of Sepharose beads with immobilized Protein A, which binds to the Fc region of immunoglobulins. This allows for the selective capture and separation of antibodies from complex mixtures.

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Lab products found in correlation

Dimethylformamide, by Merck Group (1 mentions)

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Protein A-Sepharose Protein A columns

6 protocols using protein a sepharose column

EPO-Fc Purification Using Mimetic Red 2 XL

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Example 30

Yet another alternative for purifying EPO-Fc is described herein. A mixture containing Fc, EpoFc monomer-dimer hybrid, and EpoFc dimer was applied to a Protein A Sepharose column (Amersham, Uppsala, Sweden). The mixture was eluted according to the manufacturer's instructions. The Protein A Sepharose eluate, containing the mixture was buffer exchanged into 50 mM Tris-CI (pH 8.0). The protein mixture was loaded onto an 8 mL Mimetic Red 2 XL column (ProMetic Life Sciences, Inc., Wayne, N.J.) that had been equilibrated in 50 mM Tris-CI (pH 8.0). The column was then washed with 50 mM Tris-CI (pH 8.0); 50 mM NaCl. This step removed the majority of the Fc. EpoFc monomer-dimer hybrid was specifically eluted from the column with 50 mM Tris-CI (pH 8.0); 400 mM NaCl. EpoFc dimer can be eluted and the column regenerated with 5 column volumes of 1 M NaOH. Eluted fractions from the column were analyzed by SD S-PAGE (FIG. 14).

US11168125B2. Immunoglobulin chimeric monomer-dimer hybrids (2021-11-09). BIOVERATIV THERAPEUTICS INC. [US]. Inventors: Robert T. Peters [US], Adam R. Mezo [US], Daniel S. Rivera [US], Alan J. Bitonti [US], Susan C. Low [US].

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Preparation of Anti-SRBC Polyclonal IgG

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Polyclonal IgG^a anti-SRBC was prepared from hyperimmune BALB/c serum and polyclonal IgG^b anti-SRBC from hyperimmune C57BL/6 or CB17 serum. IgG was purified on a Protein-A Sepharose column (Amersham Pharmacia Biotech, Uppsala, Sweden) [19 (link)], dialyzed against PBS, sterile filtered and stored at -20°C until use.

Bergström J.J, & Heyman B. (2015). IgG Suppresses Antibody Responses in Mice Lacking C1q, C3, Complement Receptors 1 and 2, or IgG Fc-Receptors. PLoS ONE, 10(11), e0143841.

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Preparation of IgG anti-SRBC antibodies

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Polyclonal IgG^b anti-SRBC was prepared from hyperimmune C57BL/6 serum, and polyclonal IgG^a anti-SRBC was prepared from hyperimmune BALB/c serum. IgG was purified by affinity chromatography using a Protein A Sepharose column (Amersham Pharmacia Biotech, Uppsala, Sweden) (25 (link)). Isolated IgG anti-SRBC was dialyzed against PBS, sterile filtered and stored at −20°C until use. SRBC in sterile Alsever’s solution were purchased from Håtunalab AB (Håtunaholm, Sweden) and stored at 4°C until use. SRBC were washed three times in PBS prior to use.

Bergström J.J, & Heyman B. (2017). Mice Immunized with IgG Anti-Sheep Red Blood Cells (SRBC) Together With SRBC Have a Suppressed Anti-SRBC Antibody Response but Generate Germinal Centers and Anti-IgG Antibodies in Response to the Passively Administered IgG. Frontiers in Immunology, 8, 911.

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Generating Anti-DKK1 Monoclonal Antibodies

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DKK1-P20 peptide (ALGGHPLLGV) was refolded with recombinant HLA-A2 and β₂-microglobulin (b2M) to produce DKK1-A2 monomer (MHC Tetramer Laboratory, Baylor College of Medicine, Houston, Texas, USA). Six-week-old Balb/c mice were immunized with DKK1-A2 monomer at a 2-week interval for a total of four times by an intraperitoneal injection of the antigen plus adjuvant, followed by an intraperitoneal injection of the antigen alone 3 days before harvest of splenocytes. Lymphocytes from spleens were fused with SP2/0 myeloma cells, and positive hybridomas were screened with ELISA and surface staining by flow cytometry assay. Positive clones (n=156) were isolated by limiting dilution. Large-scale antibody production of selected clones, such as C2, HMB1 and HMB7 (all are IgG1) mAbs, was achieved by intraperitoneal injection of 2×10⁶ hybridoma cells into Balb/c mice to produce ascites, followed by purifying mAbs using a protein A Sepharose column (Amersham Biosciences, Piscataway, New Jersey, USA). Mice were purchased from The Jackson Laboratory (Bar Harbor, Maine, USA). Animal care and use were approved by the Institutional Animal Care and Use Committee at MDACC (#A-3343–01) and Houston Methodist Research Institute (#A-4555–01).

Qian J., Wang Q., Xiao L., Xiong W., Xian M., Su P., Yang M., Zhang C., Li Y., Zhong L., Ganguly S., Zu Y, & Yi Q. (2024). Development of therapeutic monoclonal antibodies against DKK1 peptide-HLA-A2 complex to treat human cancers. Journal for Immunotherapy of Cancer, 12(1), e008145.

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Polyclonal IgG Anti-NP and Anti-SRBC Preparation

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Polyclonal IgG anti-NP (allotype a) and IgG anti-SRBC (allotype a) were prepared from hyperimmune BALB/c serum immunized with BSA-NP₂₀ in Freund’s complete adjuvant or 10% SRBC i.v. IgG from serum was isolated by affinity chromatography over a Protein-A Sepharose column (Amersham Pharmacia Biotech), dialyzed against PBS and stored at −20 °C. SRBC were purchased from the National Veterinary Institute. For conjugation of SRBC-NP, 4-hydroxy-3-nitrophenylacetic-e-aminocaproyl-OSu (NP-ε-Aminocaproyl-OSu, Biosearch Technologies) was dissolved in dimethylformamide (Sigma-Aldrich) at a concentration of 7.5 mg/ml. Appropriate amounts of this solution was added to 8 ml 2.5% SRBC suspensions in conjugation buffer (0.1 M NaHCO₃ with 0.15 M NaCl, pH 8.5) to a final concentration of 180, 60, or 20 μg/ml in order to achieve the coupling ratios referred to as SRBC-NP_high, SRBC-NP_int or SRBC-NP_low. The mixture was incubated for 1 h at room temperature with gentle rotation. Cells were washed 3 times in PBS and stored in PBS up to two days before use.

Xu H., Zhang L, & Heyman B. (2018). IgG-mediated immune suppression in mice is epitope specific except during high epitope density conditions. Scientific Reports, 8, 15292.

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Preparation of IgG anti-SRBC allotypes

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SRBC were obtained from Håtunalab AB (Håtunaholm, Sweden) and were stored in sterile Alsever's solution at 4°C. IgG anti-SRBC of the Ig^a and Ig^b allotypes were prepared as described (23 (link)). Briefly, BALB/c and C57BL/6 mice were immunized i.v. with 10% SRBC three times. Sera from blood obtained 4 weeks after the last immunization were run over a Protein-A Sepharose column (Amersham Pharmacia Biotech, Uppsala, Sweden). The eluted IgG fractions were dialyzed, concentrated and stored at −20° until use.

Anania J.C., Westin A, & Heyman B. (2020). IgG Suppresses Antibody Responses to Sheep Red Blood Cells in Double Knock-Out Mice Lacking Complement Factor C3 and Activating Fcγ-Receptors. Frontiers in Immunology, 11, 1404.

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