Protein A Resin and Column
Protein A is bacterial protein and can bind specifically to Fc regions of many mammalian immunoglobulins. For this reason, Protein A is conjugated onto a purification matrix called Sepharose to purify immunoglobulins, also called as antibodies, and immunoglobulin subtypes from serum, hybridoma ascites fluids, tissue culture supernatants and other biological fluids.
Protein A is expressed in E coli and the resulting recombinant protein A can be covalently conjugated onto the matrix (Sepharose 4FF). It has been well-documented that the Fc binding characteristics of naturally produced Protein A is identical to their recombinant counterparts.
The final product, the recombinant Protein A-Sepharose 4FF Premade column, is stored at 20% Ethanol stably.
rec-Protein A/Sepharose ratio：2.8~3.5mg rec-Protein A/ml Sepharose
Binding capacity：30-55mg human IgG/ml Purification matrix
22-45mg rabbit IgG/ml Purification matrix
4-12mg mouse serum/ml Purification matrix
4.5-13mg mouse ascites/ml Purification matrix
rec-Protein A molecular weight：34.85 KD
Albumin binding sites/molecule rec-protein： None
Binding Characteristics of Immunoglobulins to Protein A
• The flow rate: The column flow rate is a important parameter for purification and should be smoothly controlled. Slower flow rate can significantly increase the purification product.
• Binding Capacity: For serum samples, the total IgG concentration is 10-15mg/mL. For tissue culture supernatant or hybridoma clones, the total IgG concentration varies considerably from 2mg/ml to 18mg/ml. For samples with low IgG concentration, repeated loading of sample can increase the purification product.
Procedure for Antibody Purification
A. Additional Materials Required
• PBS Buffer: 0.1M sodium phosphate, 0.15M Sodium Chloride, pH 7.2
• Elution Buffer: IgG Elution Buffer: 0.1M glycine, pH 2.7
• Neutralization Buffer: 1M Tris (pH 9.0)
B. Immunoglobulin Purification Procedure
1. Equilibrate all reagents to room temperature before use.
2. Pack the column with resin slurry or use premade column.
3. Equilibrate column by adding the PBS buffer with a volume of 10 times related to column resin volume, and allowing the solution to drain through.
4. For sample with high concentration of Immunoglobulin, dilute sample at least 1:1 with Binding Buffer.
Note: If lipoprotein precipitation is evident in the sample of purification, centrifuge the diluted sample at 10,000 × g for 20 minutes and apply the supernatant to the equilibrated column.
5. Apply diluted sample to the column and allow it to flow completely into the resin. Do not allow the resin bed to run dry.
6. Wash the column with binding buffer, with a volume of 10 times related to column resin volume.
7. Elute antibodies with elution buffer and collect fractions. Immediately adjust eluted fractions to physiologic pH by adding 1/10 of the neutralization buffer to elute. Monitor the elution by measuring the absorbance at 280nm or by protein assay such as BCA Protein Assay Kit.
8. Pool the eluted antibody fractions that have the highest absorbance.
9. Regenerate column by washing with elution buffer. Columns can be regenerated with PBS, with a volume of 10 times related to column resin volume. Repeated tests indicate that no significant loss of binding capacities found after 10 times of repeated usage.
10. For storage, wash column with water containing 0.02% sodium azide. Store the column upright at 4°C.