HiTrap IgM Purification HP – Cytiva Colums
For 1 mL of serum sample
- 1.BUFFERS PREPARATION:
- Water and chemicals used for buffer preparation should be of high
- purity. It is recommended to filter the buffers by passing them through
- a 0.45 μm filter before use
- -BINDING BUFFER: 20mM Sodium phosphate + 0.8M Sulfato de Amonio a pH 7,5 –>para 100mL añadir 0,284g de Sodium phosphate + 10,55g Sulfato de Amonio + 100ml de agua destilada
- ELUTION BUFFER: 20mM Sodium phosphate a pH 7,5 –>para 100mL añadir 0,284g de Sodium phosphate + 100ml de agua destilada
- REGENERATION BUFFER: 20mM Sodium phosphate a pH 7,5 + 30% ISOPROPANOL —> Para 70mL elution buffer + 30 ml ISOPROPANOL
- 2. SAMPLE PREPARATION
- The sample must have the same concentration of ammonium sulphate as the binding buffer.
Gradually add small amounts of solid ammonium sulphate to the sample until the final concentration is, for example, 0.8 M. –> (Agregar 106 mg de sulfato de amonio a 1 mL de suero para obtener una concentración final de sulfato de amonio de 0,8 M)
Stir slowly and continuously during this procedure.
Pass the sample through a 0.45 μm filter immediately before loading on the column.
Note: It is important to add the ammonium sulphate gradually to avoid precipitation of IgM.
3. PURIFICATION
The recommended flow rate for HiTrap IgM Purification HP is 1 ml/min.
1 Fill the syringe or pump tubing with buffer. Remove the stopper and connect the column to the syringe (with the provided luer connector), or pump tubing, ‘‘drop to drop” to avoid introducing air into the column.
2 Remove the snap-off end at the column outlet.
3 Wash the column with 5 column volumes of each buffer: Binding buffer, elution buffer and regeneration buffer.
4 Equilibrate the column with 5 column volumes of binding buffer.
5 Apply the sample using a syringe fitted to the luer connector, or by pumping it onto the column.
6 Wash out unbound sample with 15 column volumes of binding buffer or until no material appears in the effluent.
7 Elute the IgM with 12 column volumes of elution buffer.
8 Regenerate the column with 7 column volumes of regeneration buffer.
Para 1 mL de serum, añadir 100mg de sulfato de amonio lentamente para que no precipite. Después filtrar.
Seguir protocolo de las columnas IgM purification HiTrap
Después de la purificación según el protocolo del kit de columnas, obtenemos 12mL de Eluido, que centrifugamos en tubos de Amicon – Ultra 15 (UFC900324 Milipore)
