Antibody Purification

This protocol includes an ammonium sulfate cut, affigel blue chromatography and affinity chromatography.

1. Solutions

(1) Affigel Blue Prewash

0.1 M acetic acid 5.7 ml glacial acetic acid

1.4 M NaCl 81 g NaCl

40% isopropanol 400 ml isopropanol

up to 1 liter with Q

check to make sure pH is 3.0

(2) Affigel Blue Running Buffer

10 mM K2HPO4 2.28 g K₂HPO₄

0.15 M NaCl 8.2 g NaCl

0.02% azide 0.2 g Na azide

up to 1 liter with Q

(3) 1.4 M NaCl

8.1 g NaCl

up to 100 ml with Q

(4) Saturated NH₄SO₄

767 g NH₄SO₄

add 1 liter Q

(5) 10X PBS

80 g NaCl

2 g KCl

14.4 g Na₂HPO₄

2.4 g KH₂PO₄

up to 1 liter with Q

(6) Affigel Blue Regeneration Buffer

2 M guanidine HCl or 1.5 M Na thiocyanate

(7) HiTrap Storage Buffer

10 mM Tris 7.5 1 ml 1M Tris 7.5

0.1 mM EDTA 20 ul 500 mM EDTA

50 mM NaCl 1 ml 5M NaCl

0.1% azide 0.1 g Na azide

up to 100 ml with Q

(8) 1X Coupling Buffer

0.2 M NaHCO₃ 1.68 g NaHCO₃

0.5 M NaCl 2.92 g NaCl

pH to 8.0 and bring up to 100 ml

(9) 1X Buffer A

0.5 M NaCl 2.92 g NaCl

0.01 M Tris 0.121 g Tris base

pH to 8.3 and bring up to 100 ml

Add 3.0 ml ethanolamine before use

(10) 1X Buffer A

0.1 M NaOAc 0.82 g NaOAc

0.5 M NaCl 2.92 g NaCl

pH to 4.0 and bring up to 100 ml

2. Procedure

(1) Pour a 5 ml affigel blue column (biorad) and wash with 50 ml Prewash to prep the column (when first used or if last used in more than a week).

(2) Wash with 50 ml Q, followed by 50 ml Running Buffer.

(3) Wash with 50 ml 1.4 M NaCl, if eluate is colored, then re-equilibrate.

(4) Wash with 50 ml Running Buffer.

(5) Load 1 ml serum, save flow-through, elute with 2 bed volumes running buffer (the serum albumin should stick to the column and the Ig should flow through).

(6) On ice slowly add saturated ammonium sulfate to 45% (550 ml sample 450 ml saturated ammonium sulfate). Tilt overnight at 4°C.

(7) Pellet by spinning at 3,000 rpm for 10 minutes at 4°C.

(8) Resuspend the pellet in 1 ml 1X PBS on ice (don’t vortex or agitate) and dialyze against PBS.

(9) The pharmacia HiTrap 1ml column can bind 10 micromoles of peptide or protein per ml of bed volume. Prep the HiTrap column by washing with 10 ml 50% Isopropanol, 25% Isopropanol, 10% Isopropanol, and 10 ml 1 mM ice cold HCl.

(10) Resuspend the peptide or protein in 1 ml 1X Coupling Buffer and load the column. Hold at room temperature for 1 hour.

(11) Wash with 5 ml 1X Coupling Buffer and save the flowthrough.

(12) Wash with 6 ml Buffer A, 6 ml Buffer B, and 6 ml Buffer A. Hold at room temperature for 30 minutes.

(13) Wash with 6 ml Buffer B, 6 ml Buffer A, and 6 ml Buffer B. Wash with Storage Buffer and hold at 4°C.

(14) To purify the antibody, load the affigel concentrate onto the column and hold at room temperature for 10 minutes.

(15) Wash with 50 ml 10 mM Tris 7.5 and then wash with 50 ml 10 mM Tris 7.5/500 mM NaCl. Elute with 5 ml 100 mM Glycine pH 2.5 into 1 ml 1M Tris 8.0.

(16) Dialyze and concentrate with a centricon 30.