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Protocol A.1

Antibody Purification

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

 

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

 

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 4°C.

• Pellet by spinning at 3,000 rpm for 10 minutes at 4°C.

• Resuspend the pellet in 1 ml 1X PBS on ice (don’t 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 4°C.

• 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.