HiTrap™ IgM Puriﬁcation HP 1 ml columns are prepacked with a thiophilic adsorption medium (2-mercaptopyridine coupled to Sepharose® High Performance). The binding capacity of HiTrap™ IgM Puriﬁcation HP is 5 mg/ml of medium. The column can be used for puriﬁcation of native human and human monoclonal IgM. The interaction between the protein and the ligand has been suggested to result from the combined electron donating- and accepting action of the ligand in a mixed-mode hydrophilic-hydrophobic interaction.
Protein A Sepharose® chromatography media offer an alternative solution to HiTrap™ IgM Puriﬁcation HP since some human monoclonal IgM, some IgM from normal and macroglobulinemic sera, and some monoclonal canine IgM and polyclonal IgA from pig, dog, and cat can bind to protein A.
Figure 1A shows the results from the puriﬁcation of monoclonal α-Shigella IgM from hybridoma cell culture supernatant. Analysis by SDS-PAGE (Figure 1B) demonstrated a purity level of over 80%. Results from an ELISA (not shown) indicated high activity in the puriﬁed fraction.
Figure 1. (A) Puriﬁcation of α -Shigella IgM on HiTrap™ IgM Puriﬁcation HP. (B) SDS-PAGE of sample, ﬂowthrough, and eluted pools was performed under reducing (left gel) and nonreducing (right gel) conditions. SDS-PAGE was performed on PhastSystem using PhastGel 4–15, silver staining.
Refer to Column packing and preparation for general considerations.
Binding buffer: 20 mM sodium phosphate, 800 mM ammonium sulfate, pH 7.5
Elution buffer: 20 mM sodium phosphate, pH 7.5
Wash buffer: 20 mM sodium phosphate, pH 7.5 with 30% isopropanol
The sample must have the same concentration of ammonium sulfate as in the binding buffer (0.8 M). Slowly add small amounts of solid ammonium sulfate to the sample from the hybridoma cell culture until the ﬁnal concentration is 800 mM. Stir slowly and continuously. Pass the sample through a 0.45 µm ﬁlter immediately before applying it to the column. Some monoclonal IgM might not bind to the column at a concentration of 800 mM ammonium sulfate. Binding can be improved by increasing the ammonium sulfate concentration to 1 M.
To avoid precipitation of IgM, it is important to add the ammonium sulfate slowly. An increased concentration of ammonium sulfate will cause more IgG to bind, which might be a problem if serum has been added to the cell culture medium. If there is IgG contamination of the puriﬁed IgM, the IgG can be removed by using HiTrap™ Protein A HP, HiTrap™ rProtein A FF, or HiTrap™ Protein G HP.
Ammonium sulfate can be replaced by 500 mM potassium sulfate. Most monoclonal IgM binds to the column in the presence of 500 mM potassium sulfate and the purity of IgM is comparable to the purity achieved with 800 mM ammonium sulfate.
See Affinity Purification Using HiTrap™ Columns for general instructions for puriﬁcation using HiTrap™ columns.
* 1 mL/min corresponds to approximately 30 drops/min when using a syringe with a 1 mL HiTrap™ column.
Some monoclonal IgM might bind too strongly to the column matrix for complete elution. The remaining IgM will be eluted during cleaning, but the high concentration of isopropanol will cause precipitation of IgM. Perform an immediate buffer exchange (see Column packing and preparation) or dilute the sample to preserve the IgM. Lower concentrations of isopropanol can elute the IgM and decrease the risk of precipitation.
Reuse of HiTrap™ lgM Puriﬁcation HP depends on the nature of the sample and should only be considered when processing identical samples to avoid cross-contamination.
To increase capacity, connect several HiTrap™ IgM Puriﬁcation HP columns in series. HiTrap™ columns can be used with a syringe, a peristaltic pump, or connected to a liquid chromatography system.
Store in 20% ethanol at 2 °C to 8 °C.