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1、Required Materials and Equipment

(1) 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)

(2) Ice-cold Tris-buffered saline (TBS). See recipe below

(3) 5% sodium azide solution

(4) Neutralization Buffer (NB). See recipe below

(5) Elution Buffers (pH 2.7 and pH 1.9). See recipes below

(6) Centrifuge tubes and microcentrifuge tubes

(7) Preparative centrifuge and microcentrifuge

(8) pH strips

(9) Microtiter plate reader with 600 nm filter

(10) Glass Columns

2、Guidelines for Choosing Protein A Agarose or Protein G Agarose

(1) 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

(2) 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:

Ⅰ6.06 g of Tris base

Ⅱ8.77 g of NaCl

Ⅲ 10ml of 5% sodium azide stock solution (Do not add if the antibody will used in assays of cellular response or function.)

Ⅳ 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.

(3) 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

Ⅰ12.1 g of Tris base

Ⅱ 8.7 g of NaCl

Ⅲ 200 microliters of 0.5M EDTA

Ⅳ 10 ml of 5% sodium azide stock solution (Do not add if the antibody will used in assays of cellular response or function.

Ⅴ Mix thoroughly and adjust the pH to 8.0 with 5 N HCl. Add distilled water to obtain a final volume of 100 ml. The NaCl and EDTA are added to prevent aggregation of the antibodies and loss of biological activity.