PROTOCOL FOR PROTEIN A/G PURIFICATION OF CUSTOM ANTISERUM

Affinity Purification of Immunoglobulin fractions on Protein A or Protein G based chromatography.

(Note:  Bring all buffers to room temperature before using them in the protocol)

1.        Prepare a new 1.5-2ml Protein A or Protein G column (fine mesh) in a disposable 5 ml column with top and bottom frits.  Wash column with 5 ml water twice.

2.        Equilibrate the affinity column with 5 ml of antibody binding buffer at room temperature twice.   

3.        Dilute 1:1 antiserum 10-12 ml with Antibody binding buffer (Cat # FGI-1967).  Filter the dilute antibody solution using a 0.45u syringe filter.

4.        Pass diluted antibody solution through the column and collect the pass through fraction. 

5.        Re-circulate the flow through fraction again on the same column twice.

6.        Save the flow through fraction at 4^oC (Do not freeze)

7.        Wash the column with 5 ml antibody wash buffer (Cat # FGI-1968) at room temperature.  Repeat this step 2 more times continuously monitoring the OD₂₈₀ of the flow through fraction.  Stop the washing procedure once OD₂₈₀ of the flow through fraction is at or less than 0.05

8.        Apply 5-10ml antibody elution buffer (Cat # FGI-1969) at room temperature and collect 0.5 ml fraction in tubes containing 50 ul of antibody neutralization buffer (Cat # FGI-1970) with continuous monitoring of OD₂₈₀.

9.        Wash the column with distilled water containing sodium azide (0.02%) for long-term storage.

10.     Mix all fractions gently (Do not vortex) and pool all fractions with OD₂₈₀ more than 0.1.  Mix gently the pooled fractions. 

11.     Dialyze against 2L of PBS at 4^oC over night .

12.     Measure the total protein concentration using BCA reagent (Pierce Chem. IL).

13.     Add 15% glycerol (v/v) and bovine serum albumin (0.5-1mg/ml).  If needed add preservatives (0.02% sodium azide or merthiolate)

14.     Mix well and store at -20oC for long term storage or at 4^oC for immediate use (Do not Freeze/thaw antibody sample). 

15.     Proceed for Western or ELISA determination if required.

Regeneration of Protein A/Protein G column for re-use

1.        Wash column with 5 ml distilled water containing 0.02% sodium azide. 

2.        Add 5 ml of Protein A/G regeneration buffer (Cat # FGI-1971).

3.        Wash column with 5 ml distilled water 3 times.

4.        Equilibrate the protein A/G column with 5 ml of antibody binding buffer (Cat # FGI-1967) and monitor the OD of the flow through fraction.  The OD₂₈₀ should be >0.05 less against antibody binding buffer.

5.        The column is now ready for another batch of antibody purification. 

Trouble shooting 

1.        To avoid cross-contamination of antibodies, dedicate one column of each antibody purification.

2.        If the base line OD is higher than 0.05, wash column with 5 ml Protein A/G regeneration buffer, followed by water and antibody binding buffer. 

3.        The wash step with antibody wash buffer should only start after all the diluted antibody solution is passed through the column.  Start washing by rinsing off inside of column and allowing this to flow through first before filling up the column with rest of the wash buffer. 

4.        If the temperature of elution buffer is not at room temperature the elution profile will be broader and spread over 5-10 ml volume. 

5.        If precipitation occurs in fractions with highest protein concentration, use mild antibody purification buffer (Cat # FGI-M1969).  This procedure is necessary only when antibody denaturation is problem.  Generally, the yield of purified antibody is less with this procedure.