Affinity purification of Antibodies

AFFINITY PURIFICATION OF ANTIBODIES

Last Update: December 2006

PROCEDURE - Western strip technique

NOTE: the objective of this technique is to immobilize your protein of interest on a membrane (nitrocellulose) and then use that as the ligand to purify protein-specific antibodies from crude sera

1. Run an SDS-PAGE protein gel as described

NOTE: the amount of protein loaded must be determined based on the amount of antibody to be purified. Furthermore, each antibody has its own characteristics (titer, stability, etc), wich should be taken into consideration when going through this protocol

2. Perform a Western transfer as described

NOTE: the whole gel can be use, but if the exact location of the protein of interest is known (pre-stained markers, migration positions, etc), then only that portion of the gel can be cut out and used for the transfer

3. After the transfer is complete, stain the gel for proteins (optional). Mark the position of protein markers or of the protein of interest and destain

4. Perform a Western blot as described, but stop after you have incubated with the primary antibody (sera) and performed the washes to remove any excess antibody

OPTIONAL: cut the membrane into small pieces and transfer to an eppendorf tube

5. Add enough 0.2M Glycine pH 2.5 to cover all the membrane and rock (shake) gently for 1-5 minutes (highly dependent on the stability of the antibody)

6. Transfer Glycine solution (+antibodies) to a new tube containing 0.1X volumes of 1M Tris-HCl pH 8.0

7. Immediately check that the solution has been neutralized. If not add more 1M Tris-HCl pH 8.0 until a neutral pH has been obtained

8. Aliquot and store at -20^oC until further use

PROCEDURE - GST-fusion method

A simple and reproducible method for isolation and purification of specific antibodies to a protein fused to the GST (based on Bar-Peled, M. and N.V. Raikhel. 1996. A method for isolation and purification of specific antibodies to a protein fused to the GST. Anal. Biochem. 241: 140-142)

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. 2X PBS:

Mix 200 mL of 10X PBS with 800 mL of dH₂O

4. 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

5. 0.2 M borate-NaOH pH 8.6:

Mix 200 mL 0.5 M borate-NaOH pH 8.6 with 300 mL dH₂O

6. 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

7. 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

8. 1M Tris-HCl, pH 7.5 (1 L):

Mix 121.1 g of Tris base with 800 mL of dH₂O. pH with ~60 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

17. Fresh elution buffer:

Mix 9 mg of reduce glutathione in 3 mL of 20mM Hepes-NaOH, pH 8

18. GSH-Sepaharose beads:

Pharmacia

PROCEDURE - GST-fusion method

1. Check concentration of proteins in total E.coli containing either GST alone or GST- fusion protein. Estimate amounts of GST or GST-fusion following SDS-PAGE by Coomassie stain

2. See protocol below for the preparation of E.coli proteins expressing GST and GST- fusion protein

3. Transfer 10 ml of over-night E.Coli culture each into 500 mL flask containing 90 ml of LB media and 100 ml of (100 mg/ml) ampicillin

4. Incubate at 37^oC for ~1.5-3 h at 250 rpm until A₆₀₀ reaches 0.8 or 1.0

5. Induce fusion protein expression with 0.2 mM IPTG by adding 20 mL of 1M IPTG (0.1g IPTG/0.4 mL dH₂O - filtered) to each flask. Incubate at 28^oC or 37^oC for 3-4 h at 250 rpm

6. Transfer the culture into 50 mL centrifuge tubes and centrifuge at 7,500 xg for 10 min at 4^oC to pellet the cells

7. Discard the supernatant by vacuum drawing leaving the pellet as dry as possible. Place on ice (The cells can be stored at -80^oC for several months)

8. Using a pipette resuspend the pellet completely with 10 mL of cold buffer (20 mM Hepes-NaOH, pH 7, 0.15 M NaCl, 1 mM EDTA), add 10 mL of 100 mM PMSF (phenylmethylsulfonyl - fluoride 17 mg/1 mL isopropanol) mix, and place on 4^oC

9. Lyse cells by 2 passages through a prechilled French pressure cell at 1100 psi. Cell disruption is evidenced by partial clearing of the suspension. Place on ice.

Optional: Shear DNA and lyse cells again using sonicator equipped with microprobe. Do not sonicate for more then 20 second. Avoid frothing which may denature proteins

10. Add 1 ml 20% Triton X100 to a final concentration of 1%. Mix gently for 30 min at 4^oC

11. Centrifuge at 12,000 g for 20 min at 4^oC. Transfer the clear supernatant into 15 ml polypropylene tube (The supernatant can be stored at -80^oC in 1-2 mL aliquots for several months). The supernatant is termed total soluble protein (save 500 mL for analysis = T, the rest will be loaded on glutathione-sepharose column, see below)

12. The remaining pellet i.e. inclusion bodies may contain the protein. Protein in inclusion body can be solubilized. Add to the pellet 1 mL of 20 mM Hepes-NaOH, 1 mM EDTA; 0.1 mM PMSF; 0.2 % sarcosyl. Disturb the pellet by sonication, and place the mix on a shaker for 30 to 60 min. at 4^oC

13. Centrifuge at 12,000 g for 15 min. Save the supernatant (soluble inclusion body proteins = IB)

PROCEDURE - Affinity purification of GST-fusion protein

1. Add the total soluble protein into 15 mL round bottom tube containing 1 mL of a 50% slurry of glutathione-sepharose. The resin is previously equilibrated with 30 mL 20 mM Hepes-NaOH, pH 7, 150 mM NaCl, 1 M EDTA, 0.5% Triton X-100 for 10 min, spun for 2 min at 500 xg. Discard the wash

2. Place tubes on a rocker and mix gently for 20 min at room temperature

3. Centrifuge at 500 xg for 1 min to sediment the beads. Save 0.5 mL aliquots - termed flow-through = FT, and discard the rest of the supernatant

NOTE: some % of GST-fusion protein do not bind. The use of 1 mM DTT during cell lysis may increase binding to GSH-resin

4. Wash the glutathione-sepharose beads 4 times each with ~12 mL of column equilibration solution. Mix for 2-10 min, centrifuge 1 min, and discard supernatant

5. Last Wash with 10 mL 20 mM Hepes-NaOH, leave the pellet as dry as possible. It is now ready for cross-linking. Proceed to next step

PROCEDURE - Cross linking GST-Fusion to GSH-sepahraose

1. When washing is complete, rinse the beads in 12 mL of 0.2 M borate-NaOH, pH 8.6. Centrifuge as above, aspirate off supernatant, repeat washes with borate buffer 2 more times. In the last wash leave about 1 mL of fluid above the settled beads (to 50% slurry)

2. 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. Aspirate off all supernatant

3. To cross-link the GST to GSH-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 GST-sepharose beads (final DMP conc. = 20 mM in 3 ml). Incubate for 30 min at room temperature, with continuos mixing on a rocker or by hands

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

USE DMP POWDER THAT IS DRY, AND RELATIVE NEW (~ 6 MONTHS, REFREIGERATED). BEFORE OPPENING, BRING TO ROOM-TEMP (~10 MIN) TO AVOID CONDENSATION

4. 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

5. Centrifuge at 500 xg for 2 min, aspirate of supernatant, add 10 mL of 0.2 M ethanolamine-HCl pH 8.2, mix for 1 h at room temperature

6. Centrifuge, and aspirate off supernatant. Wash the resin twice with 10 mL TBS for 2 min

NOTE: The resin can be washed twice for 3 min with 2 mL of 0.1 M glycine-HCl pH 2.5 to remove non-covalently linked molecules which may be present due to reduced coupling efficiencies. Subsequently, wash the resin with 10 ml TBS. Centrifuge, and aspirate off supernatant

7. Centrifuge, and aspirate off supernatant. Resuspend beads in 1 mL of TBS containing 0.02% sodium-azide to make ~50% slurry (v/v), and store at 4^oC

NOTE: the effectiveness of cross linking is determined at this stage. Mix the beads to a slurry and remove 20 mL of slurry. Add to slurry, that was saved before cross linking and to slurry after cross linking, 20 ml of 2X SDS-sample buffer, boil for 2 min, spin, and save the supernatant. Load the supernatant to 12% SDS-PAGE, to analyze the eluate. Boiling should release significantly less protein (GST at 26 kDa or GST-fusion at >26 kDa) from cross-linked resin

Reduced SDS-12.5% PAGE: Sample 1 -Total E.Coli expressing GST 20 mL; Sample 2 - Slurry before cross-linking; Sample 3 -Slurry after cross-linking; Sample 4 - MW protein markers; Sample 5 - Total E.Coli expressing GST-Fusion 20 mL; Sample 6 - Slurry before cross-linking; Sample 7 - Slurry after cross-linking. Stain gel with Coomassie blue.

PROCEDURE - Purification of specific antibodies to fusion protein

Pack the cross-linked GST -GSH sepharose beads or the cross-linked GST-FUSION-GSH sepharose beads at 4^oC each 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 thigh) into packed column. Prewash the packed resin with 20 mL of 50 mM 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 antibodies to antigen affinity column

Since antibody was raised against GST portion as well as the fusion protein, antiserum against GST is removed by first adsorbed against GST-affinity column. The effluent is then loaded onto GST-FUSION-affinity column in order to bind specifically antibodies that recognize the FUSION antigen. After binding, the sera is eluted with low pH, neutralize and saved

PROCEDURE - Preparation of antibodies

1. Centrifuge blood 1-2 mL (usually 30-40 mg total proteins/mL) at 8,000 xg for 10 min at 4^oC

2. Dilute the supernatant blood with to 1X PBS with 10X PBS.

3. Optional: Rabbit antiserum (2 mL) is precipitated with 50% ammonium sulfate while mixing

4. Centrifuge at 8,000 xg for 10 min at 4^oC to percipitate IgGs

5. Dissolved the percipitate IgGs with 0.1X PBS (check protein concentration)

PROCEDURE - Cross-adsorption of Anti-GST antiserum to GST-column

All steps during protein purification are performed at 4^oC

1. Just before use equilibrate 1 mL resin of GST-affinity column with 20 mL of binding 1X PBS buffer = (10mM phosphate-NaOH, pH 7, 0.1M NaCl) + 2mM PMSF

2. Load blood or IGg fraction into GST-affinity cross-linked column. Allow binding of to occur for 2-3 h or over-night by continuously recirculating it through the column at a flow rate of 10 mL/h

3. SAVE Effleuent (= E), it contains antiserum to fusion protein. Go to Next Step

The column is drained by a peristaltic pump into a beaker; the effluent is then withdrawn from the beaker and pumped baked onto the column through a separate tube. To ensure continuous circulation, it is essential that all fittings are air-tight

PROCEDURE - Adsorption of Anti-FUSIONantiserum to GST-FUSION-column

1. Just before use equilibrate 1 mL resin of GST-FUSION-affinity column with 20 mL of binding buffer 1X PBS

Optional: add detergent such as: 1% NP-40 or 0.01% Triton X-100 during binding

2. Load 10 ml of the EFLUENT (E, see above) fraction onto GST-FUSION affinity column. Allow binding of to occur at 4^oC for 2-3 h or over-night by continuously recirculating it through the column at a flow rate of 10 mL/h. SAVE Effluent, it may contains antiserum to the fusion protein (analyse the effluent = EF1 sera later)

3. The column is drained by a peristaltic pump into a beaker; the effluent is then withdrawn from the beaker and pumped baked onto the column through a separate tube. To ensure continuous circulation, it is essential that all fittings are air-tight

4. After loading, wash the column sequentially at 1 mL/min with 30 mL of each of the following solutions: (i) TBS (10mM Tris-HCl, pH, 7.5, 150mM NaCl)

Optional: add0.1 % detergent (NP-40)

NOTE: after the last wash make sure to remove most of the fluid above the setteled gel

5. Elute the bound proteins with 2 ml of either A or B solutions:

A - 50mM sodium phosphate, pH 12, 0.01% Triton X-100

B - 0.1M glycine-HCl, pH 2.5

6. Collect three 1-mL fractions into tubes preloaded with mL of cold 1M Tris-HCl, pH 6 (when using high pH for elution A) (OR 1M Tris base when using low pH for elution B)

7. As soon as each fraction volume reaches 2 mL mix content of each tube to neutralize the eluate. SAVE eluted antisera (= EAF affinity purify)

8. 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 column in TBS containing 0.02% sodium azide at 4^oC

9. Check the eluted antiserum (EAF) and EF1 fraction on western blot against pure GST and against GST-Fusion protein, at 1:100 and 1:1000 dilution