实验概要

This protocol is designed as a quick purification method for antibodies from mammalian sera, ascites, and cell culture supernatants. It should be noted that if the starting material is serum or ascites the final preparation will contain endogenous host IgG as well as specific antibodies. In general, the presence of this endogenous IgG should not interfere with assays using the antibodies. The immunoglobulin content of normal sera from several species and from monoclonal antibody sources are given in Table (193 Kb PDF).

Note: Sigma^® offers the PURE1A Kit for purification of antibodies using protein A.

主要试剂

1. Protein A Loading Buffer: 1 M potassium phosphate, pH 9.0

2. Protein G or L Loading Buffer: 0.01M phosphate buffered saline (PBS), pH 7.4 (Product No. P4417)

3. Elution Buffer: 0.1 M citric acid, pH 3.0

4. 1.5 M Tris base, for neutralization of the eluate

主要设备

1. 3 - 5 ml syringe (column sleeve), glass wool

2. Stopcock, Luer-Lock (Product No. S7396)

3. Ring stand, clamp

4. Test tubes, rack

5. Spectrophotometer, cuvets

6. pH meter

7. Transfer pipettes, bulbs

8. Beakers, stirbars and stirplate (for buffer preparation)

9. Sterile filter units (optional)

10. Sodium azide (preservative) (Product No. S2002)

实验材料

Serum, ascites or cell culture supernatant

实验步骤

Purification Protocol (for a 1 ml column)
Notes: This protocol uses a high molarity, high pH loading buffer for Protein A to enhance binding of subclasses with a weak affinity for Protein A (such as mouse IgG1). Binding to Protein G and L takes place at neutral pH, so phosphate buffered saline is used as the loading buffer. Elution of bound Ig is accomplished in a single step with a citrate buffer, pH 3, which removes all subclasses that have bound to the resin. The capacity of Protein A and Protein G for IgG from various species may be estimated from the relative affinities given in Table (71 Kb PDF). An IgG subclass with 1-2 pluses will bind at 1-5 mg IgG per ml resin. A subclass with 3-4 pluses will bind at 10-25 mg per ml resin. Protein L is able to bind all Ig classes and can bind 3-10 mg Ig per ml resin for species with 4 pluses. It is recommended that the resin to be used have at least 2 pluses for the species and Ig class of the material to be purified.

1. Prepare buffers. Buffers may be stored at 4 °C for 1-2 weeks. Filtration through sterile filter units will prolong the life of the buffers, but is not required.

2. Prepare column sleeve. Remove plunger from syringe and discard. Press a small amount of glass wool into the bottom of the syringe, enough to form a cushion about 1/2 - 1 cm thick. Attach the stopcock. Rinse with 1-2 ml loading buffer. Ensure that the glass wool cushion remains firmly in the bottom of the syringe.

3. Suspend the resin in 2 ml loading buffer by inverting and rotating the bottle. Avoid excessive shaking. Do not vortex the slurry.

4. Pour the slurry into the syringe. Allow the excess buffer to drain through, then wash the column with 5 ml loading buffer. Do not allow the resin to run completely dry.

5. If possible, estimate the amount of Ig in the serum, ascites, or supernatant to be loaded. If the amount of Ig is not known, Table (193 Kb PDF) contains approximate levels of Igs in serum from different species, in mouse ascites, and in cell culture supernatant that may be used to estimate the amount of Ig in the starting material. Based on the capacity of the resin for Ig from the species of the starting material (see Notes above) and the amount of Ig in the starting material, calculate the volume to load.

Note: These are approximate values, to be used only as an initial guide. Actual Ig concentrations in fluids and binding to the resins will vary and optimal ratios of starting material to resin must be determined experimentally.

6. Dilute serum or ascites with 3 volumes of loading buffer. Dilute supernatant with 1 volume of loading buffer.

7. Apply diluted material to column. Collect unbound fraction in a test tube or beaker and save for possible reprocessing. Wash through unbound proteins with 5 ml loading buffer per ml resin.

8. Apply Elution Buffer to column. Collect fractions equal to 1/2 column volume. Use 10 ml elution buffer per ml resin.

9. Re-equilibrate the column with 5 ml loading buffer per ml resin. Check pH of effluent to ensure that column is equilibrated at the pH of the loading buffer.

10. Assay eluate fractions for protein by absorbance at 280 nm. (If desired, a visual determination can be done using the Total Protein Kit.)

11. Pool fractions which are positive for protein. Neutralize to pH 6 - 8 with 1.5 M Tris base.

12. If desired, the unbound fraction may be re-applied to the column to recover any Ig that did not bind on the first pass, which may occur if the amount of material loaded exceeds the column capacity.

13. Dialyze into desired buffer, i.e. PBS, pH 7, at 4 °C. The volume of the dialysis buffer should be at least 20 times the volume of the protein solution. At least 2 changes of dialysis buffer, for at least 2-4 hours each, should be done to ensure complete equilibration in the dialysis buffer.

14. If no more runs are to be performed, wash the column with PBS containing 0.05 - 0.1% sodium azide. Seal the column with a stopper or Parafilm and store at 4 °C.

Note: If desired, separation of mouse and human IgG isotypes is possible from Protein A using a gradient-forming device (i.e. Product No. G6897) to create a linear pH gradient in 0.1 M citrate buffer runningfrom pH 6.5 to pH 3.0. The total gradient volume should be at least 10 times the column volume. The disadvantages of this method of elution are that it yields several peaks, each of which must be neutralized and tested, and that it results in a much more dilute product than the single step elution.