Here, we introduce this procedure by taking the coupling of ligand to Affi-Gel10 as an example. This experiment was derived from Protein Purification and Identification Lab Guide by Houzhu Zhu.
Operation method
Coupling experiments of ligands with activation media
Materials and Instruments
Phosphate buffer Affi-Gel 10 Sodium phosphate (50 mmul L) Ethanolamine Move Materials For more product details, please visit Aladdin Scientific website.
Phosphate buffer (50 mmol/L;pH7.5~8,0)
Affi-Gel 10 (Bio-Rad Laboratories)
Sodium phosphate (50 mmul/L) with NaCl (1mol/L) (PBS)
Ethanolamine (100 mmol/L)
Procedures
1) Prepare a 1~10 mg/ml protein (ligand) solution with 50 mml/L phosphate buffer (pH7.5 ~8.0) to prepare a protein (ligand) solution of 1~10 mg/ml. This protein preparation solution must be free of other amino compounds. Therefore. Tris buffer or buffers containing ammonium salts should not be used. Available buffers include phosphate buffer, MES, MOPS, and HEPES. If the protein preparation solution contains other amino compounds, dialyze them out with 0.5 mol/L phosphate buffer (pH 7.5-8.0). A small sample of protein is retained for determination of coupling efficiency.
2) Affi-Gel 10 gel is supplied as an isopropyl alcohol suspension. The isopropyl intoxicant must be removed when used to avoid causing inactivation of the ligand protein. Pour the Affi-Gel 10 suspension into a sintered slide funnel. wash the gel several times quickly with deionized water at 4°C. Do not let the gel dry out. If the gel dries out, air bubbles trapped inside the microspheres will disable some of the linking sites.
3) Add washed Affi-Gel 10 microspheres to the protein solution (approximately 1 ml of microspheres per 0.5 ml of ligand solution). Mix gently on a shaker overnight at room temperature.
4) Separate the microspheres by filtration. Retain the filtrate. Determine the protein concentration of the filtrate and the sample left from step 1 to calculate the coupling efficiency. Protein concentration can be determined by several methods. If protein concentration is determined by OD280, 0.1 mol/L HCl must be added to reduce the pH of the solution to below 6.0. Otherwise, the N-hydroxysuccinimide released during coupling will absorb at 280 nm under neutral or high pH conditions. The coupling efficiency only affects the capacity of the gel.
5) Rinse the microspheres with phosphate buffered saline solution (PBS).
6) Incubate the microspheres in 100 mmol/L ethanolamine for 4 h or overnight to seal the remaining reactive sites.
7) Rinse the microspheres with FBS. The affinity medium is now ready for use.