Purification of Antiserum or Ascites by Protein A/G Chromatography

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发布日期：2012-04-27 10:31 文章来源：丁香园
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(2) Section II. Preparation of Antiserum or Ascites for Affinity Chromatography

Throughout this section, "antiserum" refers to antiserum or ascites.

ⅠThaw the antiserum in ice water or the refrigerator overnight to prevent aggregation of proteins. Any protein aggregate that forms during thawing may be dissolved by briefly warming the thawed antiserum to 37.

ⅡAdd sodium azide to a final concentration of 0.05%, which is a 1:100 dilution of a 5% stock solution.

Ⅲ CAUTION: Since sodium azide is toxic, wear gloves and handle the stock solution with care.

Ⅳ Clarify the antiserum by centrifugation at 15,000 x g for 5 minutes at 4. This step is performed to sediment aggregates of denatured protein and lipid; it is an important step because it removes material that can foul and block the column.

Ⅴ Remove the clarified antiserum that lies between the floating lipid and the pellet. Additional filtration may be required to remove residual lipid.

(3)Section III. Affinity Chromatography Using Protein A/G Agarose

ⅠSave a 500 microliter sample of the clarified antiserum for testing along side the purified IgG because the antibody may be acid labile.

ⅡAdd the clarified antiserum to the column at a flow rate of 2 ml per minute. (Calibrate the flow rate using a 15 ml conical centrifuge tube as the receiving vessel. Adjust the stopcock on the column to give a flow of 2 ml per minute. Do not exceed a flow rate of 2 ml per minute.) Pass the antiserum through the column twice and save the flow through in case the antibody did not bind to the column.

Ⅲ Wash the column with a volume of TBS equal to 10 fold the volume of antiserum loaded on the column. For example, if 30 ml of antiserum were loaded on the column, wash with 300 ml of PBS. Save the wash in case the antibody was eluted.

Ⅳ After washing the column, collect a 20 microliter fraction and test for protein by adding it to 180 microliters of Coomassie Blue reagent to test for protein. If the sample turns blue, wash the column with an additional 50 ml of TBS and repeat the analysis for protein.

Ⅴ Prepare 1.5 ml microcentrifuge tubes for the collection of eluted antibody. Obtain one tube for each ml of antiserum plus five extra 1.5 ml tubes. Add 100 microliters of Neutralization Buffer (NB) to each tube. Note that IgG may be eluted from protein A/G agarose by a change in temperature, ionic strength, and/or pH. All of these conditions affect the binding between the charged amino acids of protein A/G and the Fc region of the IgG heavy chain. When eluting with acidic buffer, the acid must be neutralized as quickly as possible to prevent denaturation of the IgG.

Ⅵ Once the column has been washed, drain the TBS from the column bed to avoid unnecessary dilution of the Elution Buffer. Do not let the column stand dry.

Ⅶ Gently add Elution Buffer pH 2.7 at room temperature to the column. Be careful not to disrupt the column bed. Use 15 ml of Elution Buffer pH 2.7 if less than 20 ml of antiserum was loaded. Use 20 ml of Elution Buffer pH 2.7 if 20-30 ml of antiserum was loaded. Use 25 ml of Elution Buffer pH 2.7 if 30-36 ml of antiserum was loaded. Note that these volumes are guidelines and Elution Buffer muse be added until all protein has been eluted from the column.

Ⅷ Collect roughly 1 ml fractions in the tubes prepared in step 5 above. Mix each fraction immediately and place on ice before collecting the next fraction. Neutralizing the acid pH prevents denaturation of the antibodies and loss of biological activity.

Ⅸ Add 10 ml of Elution Buffer pH 1.9 at room temperature to the column; be careful not to disrupt the column bed. Collect, mix, and save fractions as described in step 8 above.

Ⅹ Remove a 20 microliter sample of each fraction and add to 180 microliters of Coomassie Blue reagent in a microtiter plate to monitor protein elution. Continue to collect fractions until the color drops to or just below background.

Ⅺ Read the microtiter plate at 600 nm and combine all fractions with an absorbance greater than or equal to 0.2 above background at 600 nm. For example, if the background is 0.18, combine all fractions with an absorbance greater than 0.38.

Ⅻ Once the absorbance has dropped below 0.2, wash the column with 200 ml of ice-cold TBS with 0.05% sodium azide.

ⅩⅢ Store the column at 2-8℃.

ⅩⅣ Use pH paper to check the pH of the combined fractions. If the pH is less than 7.0, use NB to adjust the pH to approximately 7.4.

ⅩⅤ Use the Bradford Assay with IgG as a standard, to determine the concentration of the antibody in the combined fractions. Add glycerol to 10% of the total volume if the antibody concentration of the combined fractions is less than 0.5 mg/ml.

ⅩⅥ Store the purified antibody at 2-8℃.

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