Preparation of Protein-A -Rabbit antisera-immunoaffinity sepharose column

PREPARATION OF PROTEIN-A RABBIT ANTISERA FOR AFFINITY SEPHAROSE COLUMN
	Last Update: December 2006


PREPARE SOLUTIONS
1. 10 X PBS (1M 200 mM phosphate-NaOH, pH 7.5, 1.5 M NaCl):	Mix 200 mL of 1M NaH₂PO₄ with 400 mL dH₂O, add 300 mL of filtered 5M NaCl, adjust to pH 7.5 with ~15 mL of 10M NaOH. Add dH₂O to 1 L (Autoclave)
2. 1X PBS:	Mix 100 mL of 10X PBS with 900 mL of dH₂O
3. 0.5 M borate-NaOH pH 8.6:	Dissolve 7.7 g boric-acid in 210 mL warm dH₂O, adjust to pH 8.2 when solution reaches room temperature with ~4 mL 8M NaOH. Add dH₂O to 250 mL
4. 0.2 M borate-NaOH pH 8.6:	Mix 200 mL 0.5 M borate-NaOH pH 8.6 with 300 mL dH₂O
5. 0.2 M triethanolamine-HCl pH, 8.3 (cross linking coupling buffer):	Mix 2.7 mL of triethanolamine (Sigma T1377; 7.5M) in 90 mL dH₂O. Adjust to pH 8.3 with ~0.5 mL conc. HCl. Add dH₂O to 100 mL
6. 0.2 M ethanolamine-HCl, pH 8.2 (cross linking termination buffer):	Mix 3 mL of triethanolamine (Sigma E9508; 16.7M) in 210 mL of dH₂O. Adjust to pH 8.3 with ~2 mL conc. HCl. Add dH₂O to 100 mL
7. 3M Sodium Acetate, pH 5.2:	Mix 102 g of Sodium acetate-3H₂Owith 150 mL of dH₂O. pH to 5.2 with glacial acetic acid. Add dH₂O to 250 mL (Autoclave)
8. 1M Tris-HCl, pH 7.4 (1 L):	Mix 121.1 g of Tris base with 800 mL of dH₂O. pH with ~70 mL of cHCl. Add dH₂O to 1 L
9. 1M Tris-HCl, pH 8 (1 L):	Mix 121.1 g of Tris base with 800 mL of dH₂O. pH with ~40 mL of cHCl. Add dH₂O to 1 L
10. 1M Hepes-NaOH, pH 7:	Dissolve 59.6 g of Hepes (Sigma) in 210 mL dH₂O. Adjust to pH 7 with 10M NaOH. Add dH₂O to 250 mL
11. 1M Hepes-NaOH, pH 8:	Dissolve 59.6 g of Hepes (Sigma) in 210 mL dH₂O. Adjust to pH 8 with 10M NaOH. Add dH₂O to 250 mL
12. 20 mM Hepes-NaOH pH, 7, 0.15M NaCl (1 L):	Mix 20 mL of 1M Hepes-NaOH pH 7 with 150 mL of 1M NaOH and 830 mL of dH₂O
13. 1M Dithiothreitol (DTT):	Mix 3.09 g of DTT with 20 mL of 0.01M sodium acetate pH 5.2. Filter sterlize and store at -80^oC
14. 0.5 M EDTA pH ~7.0:	Mix 186.1 g of disodium ethylenediaminetetraacetate-2H₂O with 800 mL of dH₂O. pH to 7.0 with solid NaOH (Autoclave)
15. 10X TBS (0.2 M Tris-HCl, pH 7.5, 1.5 M NaCl):	Mix 80 g of NaCl, 2 g of KCl, 30 g of Tris base, 800 mL of dH₂O, and 0.15 g of Phenol red. pH to 7.4 with HCl. Add dH₂O to 1 L (Autoclave)
16. Dimethyl pimelimidate-HCl (DMP):	Sigma D8388. MW 259.2. Store powder in air-tight conditions at 0^oC. DMP - a bifunctional reagent with 9.2A^o carbon spacer arm. DMP reacts with free amino group on antibody protein at pH >8.2. DMP must be dry and fresh. No longer than 6 months in your shelf

PROCEDURE - Protein-A sepharose preparation
1. Into 14 mL screw cap tube (Sarstedt) tube add 1 mL of Protein A Sepharose 6 MB (Pharmacia 17-0469-01). Wash with 10 Vols of 1X PBS at room temperature for 15 min while mixing on a rocker. Centrifuge for 1 min at 500 g in a swinging bucket rotor to sediment the beads
2. Aspirate of the supernatant. Washes can be repeated 1-2 more times. After the first one, most if not all the ethanol (storage solution of Protein A Speharose) should have been removed

PROCEDURE - Antibodies preparation
1. Transfer to a new 14 mL tube 3 mL of sera (containing ~30-40 mg/ml total proteins or 20 mg total IgG). Dilute to 1X PBS with 0.3 mL of 10X PBS stock, mix and place on ice
2. Centrifuge at 12,000 xg for 10 min at 4^oC. Transfer the supernatant into a new tube and check concentration of proteins in blood (serum) by Bradford assay using HSA as standard
NOTE: Serum contains ~10 mg/ml of total IgG. Use ~100 mg of total sera to bind into 1 mL of protein-A sepharose gel

PROCEDURE - Binding of antibodies to Protein-A affinity column
1. Add to the tube containing Protein A Sepharose (1 mL) 3.3 mL of crude sera in PBS buffer
2. Incubate with continuous mixing (on a rocker) for 2-4 h (to ON) at 4^oC
3. Centrifuge at 500 xg for 3 min at 4^oC. Pipette off supernatant and save in a new tube
NOTE: The supernatant still contains anti-sera that do not bind protein-A, plus some unreacted antibodies
4. Wash beads with ~8 mL PBS at 4^oC (repeat washes, centrifugation 2-4 more times)
Optional: Save 50 mL of each wash, and analyze each by SDS-PAGE to verify complete removal of 67 kDa protein (serum albumin). If albumin is still present or the gel is not white clear, it is necessary to conduct additional washes
5. When washing is complete, rinse the beads in 8 mL of 0.2M borate-NaOH, pH 8.6. Centrifuge as above, aspirate of supernatant, repeat washes with borate buffer 2 more times. In the last wash leave about 1 mL of fluid above the settled beads
6. Mix, set aside 20 mL slurry (which will be analyzed later to compare with covalently linked sera) and centrifuge the rest at 500 xg for 2 min at 4^oC
7. Aspirate off all supernatant
8. To cross-link the antibody to protein A-sepharose beads, add 15.5 mg of DMP, dimethyl pimelimidate-HCl into 2 mL of 0.2 M triethanolamine, pH 8.3 buffer and immediately pipette the solution to 1 mL of drained Protein-A-antibody resin (final DMP conc. = 20 mM). Incubate for 30 min at room temperature, with continuos mixing on a rocker
NOTE: coupling with DMP must be at pH 8.3 or higher. Check pH is above 8.3 after addition of DMP solution to beads
9. Centrifuge at 500 xg for 2 min. Save 0.5 mL supernatant and aspirate off the rest. Terminate cross linking reaction by mixing gel with 10 mL of 0.2 M ethanolamine-HCl pH 8.2 for 5 min
10. Centrifuge at x500 g for 2 min, aspirate off supernatant, add 10 mL of 0.2 M ethanolamine-HCl pH 8.2, mix for 1 h at RT
11. Centrifuge, aspirate off supernatant. Wash the resin twice with 10 mL PBS for 2 min
12. Resuspend beads in 1 mL of PBS containing 0.02% sodium-azide to make ~50 % slurry, and store at 4^oC
The column is now ready for binding of antigen
NOTE: The effectiveness of cross linking is determined at this stage. Mix the beads to a slurry remove 20 ml of slurry. Add to slurry that was saved before and to slurry after cross linking 20 m of 2X SDS-sample buffer, boil for 2 min, spin, and save the supernatant. Load the supernatant to 10% SDS-PAGE, to analyze the eluate. Boiling should release significantly less antibody (heavy chain at 55 kDa and light chain at 25 kDa) from cross-linked resin. Load on the gel 2nd and last FT after antibody binds to protein-A, as well as the FT after cross linkng reaction. The affinity column are usually stable for over 1 year when stored at 4^oC

PROCEDURE - Column technique
Pack the cross-linked antibody-protein A-sepharose beads at 4^oC into disposable plastic 10 mL column. Set up the column such that the column outlet be connected by a tube (1.3 mm inner diameter) to a peristaltic pump. Washing solution is delivered to the column from a reservoir constructed from a 60-ml syringe fitted with 16-1/2-gauge needle which is connected to the column by piercing a rubber stopper inserted (air tight) into packed column. Prewash the packed resin with 20 mL of 50mM sodium-phosphate pH 12, to remove adsorbed but non-cross-linked antibody, allowing the eluate to drain by gravity. Neutralize the resin immediately, by washing with 20 mL of 1M Tris-HCl, pH 8

PROCEDURE - Binding of antigen to antibody-resin
1. Just before use equilibrate 2 ml 50% slurry of antibody-cross linked to protein A-sepharose with 20 mL of binding buffer (10 mM Hepes-NaOH, pH 7, 0.1 M NaCl, 2 mM PMSF). Pack the resin into a 20 mL column or transfer to 15 mL centrifuge conical tube. Remove most of the solution but do not allow the gel to dry
2. Mix 10 mL of protein sample diluted or prepared in binding buffer onto affinity resin. Allow binding to occur for 1 h to over-night by continuously mixing
NOTE: When using column, seal (or cap) the column in the bottom and the top and place on a rocker
NOTE: When using batch, cap the tube and place on a rocker
3. After loading, wash the column sequentially at 1 mL/min with 30 mL of each of the following solutions:
3.1 (i) TBS (20 mM Tris-HCl, pH, 7.5. 150 mM NaCl)
3.2 or (i) Batch method: wash 2X with 10 mL of TBS
4. Wash the column with 10 ml pre-elution buffer: (ii) 0.1X TBS (2 mM Tris-HCl, pH, 7.5. 15 mM NaCl)
Optional: (iii) 0.1X TBS containing 0.1% detergent (base on affinity of antibody to antigen)
NOTE: Make sure that the last wash buffer (pre-elution buffer must have low buffering capacity compare with elution buffer)
NOTE: When the method is used to isolate intact vesicle, beware that addition of detergent may solubilized certain membrane proteins
5. Elute the bound proteins with 5 mL of 50 mM sodium phosphate, pH 12, or 0.2M Glycine-HCl, pH 2.5. Collect five 1-mL fractions into tubes preloaded with 1 mL of cold 1M Tris-HCl, pH 6 or 60 mL of 1M Tris-HCl, pH 8. As soon as each fraction volume reaches 2 mL mix content of each tube to neutralize the eluate, and place on ice
Batch: add 5 mL of 0.2M Glycine-HCl, pH 2.5, mix for 5-10 minutes, collect in a new tube containing 60 mL of 1M Tris-HCl, pH 8
6. Regenerate the column immediately after use by washing the column with 40 mL of 1M Tris-HCl, pH, 8. The column can be reused at least 10-times. Store the column in TBS containing 0.02% sodium azide at 4 ^oC.