提取IgG的具体步骤-英文.doc

Purification of Antiserum or Ascites by Protein A/G Chromatography Required Materials and Equipment Protein A or G agarose gel column (10 ml or 5 ml of packed beads; see guidelines below for choice of protein A or protein G) Ice-cold Tris-buffered saline (TBS). See recipe below. 5% sodium azide solution Neutralization Buffer (NB). See recipe below. Elution Buffers (pH 2.7 and pH 1.9). See recipes below. Centrifuge tubes and microcentrifuge tubes Preparative centrifuge and microcentrifuge pH strips Microtiter plate reader with 600 nm filter Glass Columns Guidelines for Choosing Protein A Agarose or Protein G Agarose Antibodies bind with different affinities to protein A and protein G conjugated agaroses. Use the chart below to choose the best affinity agarose for your antibody Species and Type of Antibody Agarose Rabbit IgG Protein A or Protein G Mouse IgG1 Protein G Mouse IgG2 Protein A or Protein G Mouse IgG3 Protein G Sheep IgG Protein G but binds weakly Rat IgG Protein G but binds weakly Guinea Pig IgG Protein A Dog IgG Protein A Goat IgG Protein G Pig IgG Protein A Buffer Preparation To prepare 1 liter of TBS (50 mM Tris-HCl, pH 7.4; 150 mM NaCl; 0.05% sodium azide) add the following to 800ml of distilled water: 6.06 g of Tris base 8.77 g of NaCl 10ml of 5% sodium azide stock solution (Do not add if the antibody will used in assays of cellular response or function.) Mix well and adjust the pH to 7.4 with 5 N HCl. Bring the volume up to 1 L with distilled water and recheck the pH after chilling on ice. To prepare 100 ml of NB (1 M Tris-HCl, pH 8.0; 1.5 M NaCl; 1mM EDTA; 0.5% sodium azide), add the following to 80 ml of distilled water: 12.1 g of Tris base 8.7 g of NaCl 200 microliters of 0.5M EDTA 10 ml of 5% sodium azide stock solution (Do not add if the antibody will used in assays of cellular response or function.) Mix thoroughly and adjust the pH to 8.0 with 5 N HCl. Add distilled water to obtain a final