Immunoaffinity purification
From Diseasepedia.net
No6. Immunoaffinity purificationSummary
Purification factor is 1,000-to 10,000-fold
Rapid antigen purification, columns often reusable
Yields purified antigen
Not useful for quantitation
Efficiently depends on antigen concentration and antibody affinity
Needs moderate affinity antibodyeral, lower-affinity antibody may work
Caution
DMP, ethanolamine, SDS
1. Bine the antibody tp protein A or protein G beads. For general-purpose columns, bind approximately 2 mg of monoclonal antibody or affinity-purified polyclonal antibodies per milliliter of wet beads. Mix the antibodies and protein A or G beads together in a loose slurry, using about 1 ml of bead volume per 10 ml of total solution. Incubate at room temperature for 1 hour with gentle rocking.
2. Wash the beads twice with 10 volumes of 0.2M sodium borate (pH 9.0) by centrifugation at 3000g for 2 minutes or 10,000g for 30 seconds.
3. Resuspend the beads in 10 volumes of 0.2 M sodium borate (pH 9.0) and remove and place aside the equivalent of 10 ul of wet bead volume. Add enough dimethyl pimelimidate (solid) to the total bead slurr to bring the final concentration to 20 mM.
4. Incubate for 30 minutes at room temperature with gentle mixing. Remove the equivalent of 10 ul of the coupled beads.
5. Stop the reaction by washing the beads once in 0.2M ethanolamine (pH 8.0) and then incubate for 2 hours at room temperature in 0.2M ethanolamine with gentle mixing. The beads can be stored at this stage by washing in PBS and sorting in 0.01% Merthiolate in PBS.
6. Checj the efficiently of coupling by boiling samples of beads taken before and after coupling in Laemmli sample buffer. Run the equivalent of 1 ul and 9 ul of both samples on a 10% SDS-polyacrylamide gel and stain with Coomassie blue. Good coupling is indicated by heavy-chain bands (55,000 MW) in the "before" but not in the "after" lanes.
7. Transfer the antibody beads to a suitable column. Rinse the mixing chamber with PBS to collect the remaining beads. If possible, use only as much antibody-bead matrix as needed to bind the total amount of antigen in the preparation.
8. Wash the column with 20 bed-volumes of buffer used to prepare the antigen.
9. Apply the antigen solution to the column. Pass the solution through the column with a flow rate of approximately 1 ml/hour per each 1 ml of column volume. This is easiest to control using a pump.
10. Wash the column with 20 bed-volumes of binding buffer.
11. Change the buffer in the column to the pre-elution buffer by washing with 20 column bed-volumes.
12. Using a stepwise elution, sequentially pass 0.5 bed-volume of the elution buffer through the column. Collect each fraction in seperate tubes. Ig either high or low pH is used to elute the column, the collection tubes should contain 0.1 bed-volume of neutralizing buffer.
13. Check each tube for the presence of the antigen. Combine tubes with high concentrations. Depending on how the antigen will be used, it may be necessary to dialyze the resulting eluted proteins to change the buffer.
14. Return the column to the starting buffer by passing 20 column volumes through the matrix. Add 0.01% Methiolate for long-term storage(4°C).
