IgA was isolated first from plasma using peptide M agarose beads (InvivoGen #GEL-PDM). The pass-through plasma was enriched sequentially for IgG using protein G agarose beads (InvivoGen #GEL-AGG) and for IgM using a HiTrap IgM column (G.E. Healthcare #17-5110-01). An additional step was performed using Protein A Plus mini-spin columns to separate IgG from IgM. Protein concentrations were determined with Nanodrop (Thermo Scientific).
Hitrap igm column
Manufactured by GE Healthcare
HiTrap IgM columns are affinity chromatography columns designed for the rapid and efficient purification of immunoglobulin M (IgM) antibodies from biological samples. The columns utilize a specialized ligand that selectively binds to IgM, allowing for the capture and isolation of this antibody class from complex mixtures.
Automatically generated - may contain errors
Lab products found in correlation
Hitrap, by Cytiva (7 mentions)
Protein g sepharose beads, by Thermo Fisher Scientific (3 mentions)
Nanodrop, by Thermo Fisher Scientific (1 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Protein g sepharose bead, by GE Healthcare (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Polyethylenimine (pei), by Polysciences (1 mentions)
Kolliphor p188, by Merck Group (1 mentions)
Sepharose g beads, by Thermo Fisher Scientific (1 mentions)
8 protocols using hitrap igm column
1
Immunoglobulin Fractionation from Plasma
2
Purification and Quantification of Immunoglobulins
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IgA was isolated first from plasma using peptide M agarose beads (InvivoGen; GEL-PDM). The pass-through plasma was enriched sequentially for IgG using protein G agarose beads (InvivoGen; GEL-AGG) and for IgM using a HiTrap IgM column (GE Healthcare; no. 17-5110-01). An additional step was performed using Protein A Plus mini-spin columns to separate IgG from IgM. Protein concentrations were determined with a NanoDrop spectrophotometer (Thermo Scientific).
3
Isolation and Characterization of Insulin-Specific Immunoglobulins
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Serum of immunized mice was collected immediately after euthanization. Removal of antigen bound to antibodies was achieved by repeated freeze-thaw cycles of the serum and pH-shift during elution (23 (link)). Protein G sepharose beads (ThermoFisher) were used according to the manufacturers protocol and dialyzed overnight in 10 times sample volume in 1x PBS to isolate IgG. For IgM, HiTrap IgM columns (GE Healthcare, Sigma-Aldrich) were used according to the manufacturers protocol and dialyzed overnight in 10 times sample volume 1 x PBS. Quality-control of the isolated immunoglobulins was done via SDS-PAGE and Coomassie-staining and the concentration of insulin-specific immunoglobulins determined via ELISA.
4
Isolation of Antigen-Specific Antibodies
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The serum of injected mice was collected immediately after euthanasia at the indicated days. The antigen bound to antibodies was removed by repeated freeze–thaw cycles of the serum and pH shift during elution (71 (link)). Protein G Sepharose beads (Thermo Fisher) were used according to the manufacturer’s protocol and obtained antibodies dialyzed overnight in 10 times sample volume of 1× PBS. For isolation of IgM, HiTrap IgM columns (GE Healthcare, Sigma-Aldrich) were used according to the manufacturer’s protocol and dialyzed overnight in 10 times sample volume of 1× PBS. The quality of the isolated immunoglobulins was checked via SDS-PAGE and Coomassie staining, and the concentration of antigen-specific immunoglobulins was determined by ELISA.
5
IgM Purification from Human Serum
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For IgM purification from human serum, IgG depletion was performed by incubating the samples with Protein G Sepharose beads (GE Healthcare, Sigma-Aldrich) according to manufacturer’s instructions.
For IgM purification from IgG-depleted human serum and from HEK293-6E cell supernatant, HiTrap® IgM columns (GE Healthcare, Sigma-Aldrich) were used according to the manufacturer’s protocol and eluates were dialyzed overnight in 300-fold sample volume 1x PBS. Quality control of the isolated immunoglobulins was addressed via SDS–PAGE stained with Coomassie-brilliant blue R-250 (BIO-RAD) and the quantification of eluted proteins was assessed via ELISA.
For IgM purification from IgG-depleted human serum and from HEK293-6E cell supernatant, HiTrap® IgM columns (GE Healthcare, Sigma-Aldrich) were used according to the manufacturer’s protocol and eluates were dialyzed overnight in 300-fold sample volume 1x PBS. Quality control of the isolated immunoglobulins was addressed via SDS–PAGE stained with Coomassie-brilliant blue R-250 (BIO-RAD) and the quantification of eluted proteins was assessed via ELISA.
6
Transient Transfection of HEK293-6E Cells
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HEK293-6E cells were cultured in FreeStyle F17 expression media (Invitrogen) supplemented with 0,1% Kolliphor ® P188 (Sigma-Aldrich) and 4mM L-Glutamine (Gibco Life Technologies). Transfections were performed according to the manufacturer’s instructions. Briefly, cells were transfected using Polyethylenimine (Polysciences) with two pTT5 plasmids encoding heavy and light chain of the antibody of interest (total 1 µg DNA/ml of culture). 24-48 hours post transfection cells were fed with Tryptone N1 (TekniScience Inc #19553) to a final concentration of 0,5%.
Harvesting was performed 120 hours post transfection. Antibodies were purified using HiTrap® IgM columns (GE Healthcare, Sigma-Aldrich) as described below.
Harvesting was performed 120 hours post transfection. Antibodies were purified using HiTrap® IgM columns (GE Healthcare, Sigma-Aldrich) as described below.
7
Purification and Quantification of Insulin-Specific Ig
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Sera of immunized mice were taken immediately after euthanasia and either IgM or IgG were purified. Removal of antigen bound to antibodies was achieved by repeated freeze‐thaw cycles of the serum and pH‐shift during elution (Reverberi & Reverberi, 2007 (link)). For IgG protein G sepharose beads (Thermo Fisher) were used according to the manufacturer’s protocol and dialyzed overnight in 10 times sample volume in 1× PBS. For IgM, HiTrap IgM columns (GE Healthcare, Sigma‐Aldrich) were used according to the manufacturer’s protocol and dialyzed overnight in 10 times sample volume 1× PBS. Quality control of the isolated immunoglobulins was addressed via SDS–PAGE and Coomassie and the amount of insulin‐specific immunoglobulins determined via ELISA. Finally, 20–50 µg (1–10 µg insulin‐specific‐Ig) were injected intravenously.
8
Isolation of Antigen-Specific Immunoglobulins
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Serum of injected mice was collected immediately after euthanization at indicated days. Antigen bound to antibodies was removed by repeated freeze-thaw cycles of the serum and pH-shift during elution (Reverberi & Reverberi, 2007) . Protein G sepharose beads (ThermoFisher) were used according to the manufacturers protocol and obtained antibodies dialyzed overnight in 10 times sample volume of 1x PBS. For isolation of IgM, HiTrap IgM columns (GE Healthcare, Sigma-Aldrich) were used according to the manufacturers protocol and dialyzed overnight in 10 times sample volume of 1 x PBS. The quality of the isolated immunoglobulins was checked via SDS page and Coomassie-staining and the concentration of antigen-specific immunoglobulins determined by ELISA.
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