Human IgM Immobilization kit (Code : hIgM-FF KIT)
(for fabrication of 3 columns of 1 ml of hIgM-SepharoseTM fast flow)
- Human IgM Activated Binding Gel (Code : hIgM-AFF) : 3 x 1 ml gel column.
Matrix : SepharoseTM fast flow
Binding capacity > 10 mg hIgM per ml of wet gel.
Maximum pressure : 3 bars (43 psi, 0.3 MPa).
Human IgM Activated Binding Gel recognizes hIgM at Fc portion where the coupling with the gel will be occured.
Shelf life of the gel : 4 years is stored at 4°C without loss of binding capacity.
- Human IgM Binding & Coupling Buffer (Code : hIgM-BCB) : 100 ml.
- Human IgM Saturation Reagent (Code : hIgM-SR) : 50 ml.
- Immuno Acid Washing Buffer (Code : IAf-WA) : 125 ml.
- Immuno Basic Washing Buffer (Code : IAf-WB) : 125 ml.
INSTRUCTION FOR USE
All operations are carried out at room temperature and gravity flow rate.
I. SYNTHESIS OF IMMUNOAFFINITY COLUMN
- Equilibrate the column hIgM-AFF (Human IgM Activated Binding Gel) with 4 ml of Human IgM Binding & Coupling Buffer (hIgM-BCB).
- Make up the sample with the purified hIgM (maximum 5 mg for monoclonal and 10 mg for polyclonal IgG) dissolved in the Human IgM Binding and Coupling Buffer (hIgM-BCB), at a concentration of 1-2 mg/ml.
- - Apply the sample prepared in point 2. into the column prepared in point 1. at a flow rate of approx. 20 ml/cm2/hour.
- Reload the flow through into the column at the same flow rate.
- Repeat the same once more.
- Wash the column with 20 ml of Human IgM Binding & Coupling Buffer (hIgM-BCB)
- Load into the column 15 ml of Human IgM Saturation Reagent (Code : hIgM-SR) at a flow rate of approx. 20 ml/cm2/hour.
- Wash the column with
- 20 ml of Immuno Acid Washing Buffer (Code : IAf-WA).
- then with 20 ml of Immuno Basic Washing Buffer (Code : IAf-WB).
- The column is now ready for use. This immunoaffinity column can be re-used 10 times without loss of binding capacity for the corresponding antigen.
- Storage : at 4°C in 1PBS or 2TBS buffer pH 7.4 in the presence of NaN3 0.1% (w/v).
1PBS : Phosphate buffered saline (K2HPO4 50mM, NaCl 150mM).
2TBS : Tris buffered saline (Tris.HCl 50mM, NaCl 150mM).
It is not difficult to immobilize a protein on a solid support using any classic activation method such as the cyanogen bromide or N-hydroxysuccinimidylcarboxylate... However, if the protein of interest is a specific antibody, its biological activity will be affected if not entirely lost after the covalent linkage between the solid support with the active site H2N of the antibody.
AFFILAND has developed an Antibody "Activated Binding Gel" for immobilizing the antibody at its Fc portion leaving free its Fab. The biological properties of immobilized antibody will be so conserved.
II. PURIFICATION OF ANTIGEN BY CORRESPONDING IMMUNOAFFINITY COLUMN
The Protocol will be provided with the order.
IgG & IgM & IgY Immobilization Kits