| Contributor: |
The Laboratory of Donald Rio at the University of California, Berkeley
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1. Transfer 0.5 ml of peptide-coupled CH-sepharose beads to an econocolumn with a Pasteur pipette.
2. Wash column by running 10 column volumes (5 ml) of PBS through the column to equilibrate it.
3. Transfer the beads to a microcentrifuge tube (or a 15 ml conical tube) with 0.5 to 3 ml of ascites fluid or serum.
4. Wash out the column with 1 ml of Buffer B to flush the residual beads into the tube.
5. Rock the tube containing the beads and serum/ascites at 4°C overnight.
6. Centrifuge the tube in a Beckman Accuspin at 500 rpm for 5 min at 4°C.
7. Put the tube contents back into the column and save the flow-through at 4°C.
8. Run 2 column volumes of Buffer B through the column followed by 2 column volumes of Buffer B/LiCl.
9. Run 4 volumes of Buffer B through the column.
10. Finally, run 2 column volumes of PBS through the column. Repeat this PBS wash until no more NP-40 elutes from the column (check for protein in eluate by the Bradford assay).
11. Determine how much 1.0 M Tris, pH 8.0 is required to neutralize a fraction volume (250 μl) of Elution Buffer (this usually requires about 15 to 25 μl of 0.1 M Tris, pH 8.0). Put this amount of 1.0 M Tris, pH 8.0 into a series of microcentrifuge tubes that will be used to collect fractions of column eluate (see Hint #2).
12. Run Elution Buffer through the column to elute the antibody and collect 10 drop (250 μl) fractions in the microcentrifuge tubes.
13. Find the peak and side antibody-containing fractions by performing a Bradford assay on aliquots of the fractions.
14. Pool the peak and side fractions (see Hint #3).
15. Add RIA-grade Bovine Serum Albumin (BSA) to the pooled fractions to a final concentration of 10 μg/ml and store the affinity-purified antibodies at 4°C.
16. Reequilibrate the column by running PBS through the column until the flow-through reaches a pH of about 7.0. Store the column in PBS/Azide at 4°C.
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| PBS |
| pH 7.2
2.7 mM KCl
4.3 mM Sodium Phosphate Dibasic (Na2HPO4)
1.8 mM Potassium Phosphate Monobasic (KH2PO4)
137 mM NaCl
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| Buffer B/LiCl |
| 1.0 M LiCl
50 mM Tris, pH 8.0
0.5% (v/v) NP-40
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| 1.0 M TRIS, pH 8.0 |
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| Buffer B |
| 120 mM NaCl
50 mM Tris, pH 8.0
0.5% (v/v) NP-40
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| Elution Buffer |
| 150 mM NaCl
pH 2.5
50 mM Glycine
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| PBS/Azide |
| 0.02 % (w/v) Sodium Azide (NaN3; CAUTION! see Hint #1)
pH 7.2
1.8 mM KH2PO4
4.3 mM Na2HPO4
2.7 mM KCl
137 mM NaCl
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Sodium Phosphate, Dibasic
Bovine serum albumin, RIA-grade
Tris
Sodium Azide
Potassium Chloride
Sodium Chloride
NP-40
Glycine
Lithium Chloride
Potassium Phosphate, Monobasic
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1. CAUTION! This substance is a biohazard. Consult this agent's MSDS for proper handling instructions.
2. The pH of the eluate will be neutralized immediately by the 1.0 M Tris, pH 8.0.
3. Just because there is no protein detectable by the Bradford assay in a fraction, does not mean that there is no antibody present. Check fractions by PAGE followed by Coomassie blue staining if the Bradford is negative (see protocols for PAGE and Coomassie Blue Staining).
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