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Monday, May 03, 2004
Description This protocol includes an ammonium sulfate cut, affigel blue chromatography and affinity chromatography Procedure 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). Wash with 50 ml Q, followed by 50 ml Running Buffer. Wash with 50 ml 1.4 M NaCl, if eluate is colored, then re-equilibrate. Wash with 50 ml Running Buffer. 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). On ice slowly add saturated ammonium sulfate to 45% (550 ml sample + 450 ml saturated ammonium sulfate). Tilt overnight at 4C. Pellet by spinning at 3,000 rpm for 10 minutes at 4C. Resuspend the pellet in 1 ml 1X PBS on ice (dont vortex or agitate) and dialyze against PBS. 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. Resuspend the peptide or protein in 1 ml 1X Coupling Buffer and load the column. Hold at room temperature for 1 hour. Wash with 5 ml 1X Coupling Buffer and save the flowthrough. Wash with 6 ml Buffer A, 6 ml Buffer B, and 6 ml Buffer A. Hold at room temperature for 30 minutes. Wash with 6 ml Buffer B, 6 ml Buffer A, and 6 ml Buffer B. Wash with Storage Buffer and hold at 4C. To purify the antibody, load the affigel concentrate onto the column and hold at room temperature for 10 minutes. 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. Dialyze and concentrate with a centricon 30. Recipes Solutions 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 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 1.4 M NaCl 8.1 g NaCl up to 100 ml with Q Saturated NH4SO4 767 g NH4SO4 add 1 liter Q 10X PBS 80 g NaCl 2 g KCl 14.4 g Na2HPO4 2.4 g KH2PO4 up to 1 liter with Q Affigel Blue Regeneration Buffer 2 M guanidine HCl or 1.5 M Na thiocyanate 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 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 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 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 Supplies Tips (责任编辑：泉水) |
Antibody Purification
时间:2005-07-18 00:00来源:Scienceboard.net 作者:admin 点击:
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