IgM isolation and subsequent purification of serum samples was carried out using the ÄKTA™ Pure system (GE Healthcare, UK) at 4°C, using prepacked HiTrap™ IgM Purification HP column as the stationary phase. IgM containing fractions were pooled; protein purity was assessed by SDS-PAGE and subjected to fluorometric quantitation using Qubit (ThermoFisher Scientific, UK).
Hitrap igm purification hp column
Manufactured by GE Healthcare
Sourced in United States
The HiTrap™ IgM Purification HP column is a prepacked column designed for the purification of immunoglobulin M (IgM) from biological samples. The column utilizes a specialized resin that enables the efficient capture and separation of IgM from other molecules present in the sample.
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Lab products found in correlation
Nanodrop, by Thermo Fisher Scientific (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Kta pure system, by GE Healthcare (1 mentions)
Profinia protein purification system, by Bio-Rad (1 mentions)
Cd45 apcefluor780 clone 30 f11, by Thermo Fisher Scientific (1 mentions)
Rat anti mouse cd16 cd32 monoclonal antibody, by Thermo Fisher Scientific (1 mentions)
Facscanto flow cytometer, by BD (1 mentions)
4 protocols using hitrap igm purification hp column
1
Serum IgM Purification and Characterization
2
Monoclonal Antibody Purification and Characterization
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The isotype of each mAb was determined using the PierceTM Rapid ELISA Mouse mAb Isotyping Kit according to the manufacturer’s instructions. IgG mAbs were purified from 100 ml of high-density hybridoma culture supernatant (7–10 days stationary batch) by automated affinity chromatography using 1 ml bed volume Mini Affi-Prep® Protein A cartridges. Briefly, hybridoma culture supernatants were first concentrated 10-fold using centrifugal protein concentrators (Sartorius, Germany), then diluted with PierceTM Protein A IgG binding buffer (1:4 ratio) and filtered through a 0.22-μm filter before being purified on the ProfiniaTM Protein Purification System (Bio-Rad, USA) under the default program settings of the Protein A/G affinity method. The mAbs were eluted using PierceTM IgG elution buffer. In the case of IgM mAbs, hybridoma culture supernatant was purified using HiTrap IgM purification HP column according to the manufacturer’s instructions (GE Life Sciences, USA). Concentration of the purified mAbs was determined using the PierceTM BCA Protein Assay Kit according to the manufacturer’s instructions. The purity of the mAbs was assessed by 16% SDS-polyacrylamide gel analysis.
3
Purification of Anti-Hepatitis A IgM
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4
Leukocyte Profiling in Late-Stage STO Tumors
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Leukocyte population was evaluated in peritoneal washing and spleen cell suspension obtained from mice bearing late-stage STO tumors, treated or not with CpG-ODN. Briefly, after red blood cells lysis, cells were stained (30 min at 4 °C) with the following antibodies: CD45APCeFluor780 (clone 30-F11; eBioscience, San Diego, CA, USA); CD49bFITC (clone DX5; Miltenyi, Bergisch Gladbach, Germany); CD11bPECy5 (clone M1/70; eBioscience); Ly6G/GR-1PE (clone RB6-8C5; Southern Biotech, Birmingham, AL, USA); F4/80PerCPCy5.5 (clone BM8; eBioscience); CD11cPECy7 (clone N418; eBioscience); CD11bPerCPCy5.5 (clone M1/70; eBioscience); CD5PEVio770 (clone 53-7.3; Miltenyi); CD23FITC (clone B3B4; Miltenyi); CD19APC (clone 6D5, Miltenyi). Rat anti-mouse CD16/CD32 monoclonal antibody (eBiosciences) was used to prevent nonspecific binding to mouse Fc receptors. Cells were examined by FACSCanto flow cytometer (Becton–Dickinson, San Jose, CA, USA) and data analyzed using FlowJo software (TreeStar Inc., Ashland, OR, USA). Analyses were performed gating on CD45 + cells after doublet exclusion.
Moreover, IgM were purified from peritoneal lavages by affinity chromatography on HiTrap IgM Purification HP column (GE HealthCare, Uppsala, Sweden). The concentration of purified IgM was determined by Pierce BCA Protein Assay Kit (Thermo Scientific, Waltham, MA USA).
Moreover, IgM were purified from peritoneal lavages by affinity chromatography on HiTrap IgM Purification HP column (GE HealthCare, Uppsala, Sweden). The concentration of purified IgM was determined by Pierce BCA Protein Assay Kit (Thermo Scientific, Waltham, MA USA).
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