Affinity-Purification PROTOCOL
Affinity purification is a method for purifying immunoglobulins based on the reversible binding of antigen and antibody. By cross-linking an antigen to a resin, an antibody that specifically reacts with the antigen is purified, and this purification method removes a large number of non-specific immunoglobulin components, resulting in an antibody with higher specificity.
Preparation of antigen affinity columns
I. Materials and Reagents
l CNBr-activated Sepharase 4B, GE Healthcare
l 1mM HCL
l 1L Buffer Ligation Solution
100 mM NaHCO3 8.4 g, 500 mM NaCl 29.2 g, and add ddH2O to 1 L.
l Sealing Solution: 0.5M ethanolamine, 0.5M NaCl pH 8.0
l pH 4.0 Wash Solution:
0.5M NaCl 19.22 g. Add ddH2O to 1 L, add acetic acid to adjust pH to 4.0.
l pH 8.0 washing solution:
0.1M Tris 121.1 g, add ddH2O to 1 L, add hydrochloric acid to adjust PH to 8.0
l PBS buffer:
10 mM Na2HPO4-12H2O 3.58 g, 1.8 mM KH2PO4 0.24 g, 137 mM NaCl 8 g, 2.7 mM KCl 0.2 g, pH 7.4, make up to 1 L with ddH2O.
II. Operational Process
1. Pretreatment of CNBr-activated Sepharase 4B Weigh appropriate amount of CNBr-activated Sepharase 4B dry powder (1mg lyophilized powder can yield 3.5ml beads, typically 1ml beads can bind 5-10mg of protein), place in pre-cooled 1mM HCL beaker and place at 4 degrees for 15 minutes. An equal volume of beads was added to the affinity purification column and washed as 1ml beads 200ml 1mM HCL. The remaining HCL was then removed.
2. The solubilized proteins or peptides were added to the appropriate amount of coupling buffer and then injected into the purification column with beads. The column was incubated simultaneously under two conditions: 3-4 hours at room temperature and overnight at 4 degrees Celsius.
3. At the end of incubation, wash the attached beads three times with coupling buffer, then add blocking solution, 1 ml beads with 8 ml blocking solution. Incubate overnight at 4°C or for 4 hours at room temperature.
4. Drain the blocking solution, wash 3 times with coupling buffer, then wash 3 times with PH4.0 and PH8.0 alternating acid and base.
5. The washed beads were added to 20% ethanol in PBS and stored at 4 degrees.
Affinity purification step
I. Materials and Reagents
l PBS buffer:
19.38 mM Na2HPO4-12H2O 69.4 g, 1.8 mM KH2PO4 2.4 g, 121.24 mM NaCl 70.9 g. Adjust pH to 7.84, then condense to 10 L.
l Phosphoric acid neutralizing solution:
Na2HPO4-12H2O 322.3 g, KH2PO4 4.9 g. Make up to 1 L with water.
l Eluent:
150 mM NaCl 87.6 g. Adjust pH to 2.0-2.5 with HCl, then dilute to 10 L.
l Kaomas Brilliant Blue: 100mg Kaomas G-250, 50ml ethanol, 100ml phosphoric acid.
II. Operational Process
1. After thawing at 4°C, place the serum in a 56°C water bath for 30 minutes, ensuring that the serum level is within the water bath.
2. Centrifuge the serum at 4°C, 3500 rpm for 20 minutes.
3. The lipids on the surface of the centrifuged serum were aspirated with a pipette, the supernatant was collected, and a small sample of 30-50ul of serum was retained for ELISA.
4. Incubate the prepared column and serum for 1.5 hours at room temperature or in a 30°C water bath if the room temperature is low.
5. After incubation, wash 3 times with PBS containing 10 times the volume of the column.
6. Elute: Pre-elute 5-10 ml with 150 mM glycine at pH 5.0, then elute with pre-cooled pH 2.5 eluent, collect 1 ml at a time and add 50 ul of neutralization solution to the EP tube or 96-well collection plate.
7. The collection solution is used to detect the collection peaks with a Koemas assay, 3.3 ul of collection solution is added to 100 ul of Koemas, and the antibodies that are directly collected and those that need to be concentrated are judged according to the collection peaks and stored separately.
8. The collection was stopped when no color appeared in the Kaumas assay, washed with 10 ml PBS, and the incubation and elution were repeated until no collection peak appeared. A 50 ul FT sample was retained for ELISA.
9. Concentrate the collected low level antibody with PEG or ProA.
10. Store the antibody until needed.