Antibody purification is possible because the cepharose beads are conjugated with protein which has an affinity under certain conditions to immunoglobulins. Following, you'll find a basic protocol for antibody purification either by Protein A or Protein G. The purification steps for A or G are the same, however the buffers differ. 1mL of protein A will bind 20mg of antibody and 1mL of protein G will bind 11mg. The facility does offer antibody purification as a service

Protein A Buffers 1M Tris pH 8.0 20mM Tris pH 8.0 0.1M glycine pH 2.5 20mM glycine pH 5.0 1x PBS 0.1M Sodium Acetate/.5M NaCl

Protein G Buffers 1M Tris pH8.0 0.1M Sodium Acetate pH 5.0 0.1M glycine pH 2.5 0.1M Sodium Acetate/.5M NaCl

 

Protocol:

• Load protein A or G slurry into a clean column
• Sterilize with 50% Ethanol
• Wash Column with 50mL Sterile PBS
• Adjust the culture supernatent to pH 5.0 for Protein G and pH 8.0 for Protein A
• Equilibrate column with 10 column Volumes of 0.1M sodium acetate pH 5.0 (protein G) or 20mM Tris pH 8.0 (protein A)
• Load the culture supernatent and save flowthrough
• Wash column with 6 column volumes of 20mM Tris pH 8.0 (Protein A only)
• Wash column with 10 column volumes of 0.1M Sodium Acetate (protein G) or 20mM glycine pH 5.0 (protein A)
• Add 100uL of 1M Tris pH 8.0 to 10-15 labelled polypropylene tubes for elution
• Elute antibody off the column with 3-5 column volumes of 0.1M glycine pH 2.5 (both Protein A and G) at a rate of 1mL/tube
• Strip any excess antibody from the column by running 10column volumes of 0.1M Sodium Acetate/0.5M NaCl stripping buffer.
• Make a 1:10 dilution of the eluted product (50uL sample + 450uL 1xPBS)
• Read samples on a spectrophotometer at 280nm against a 1xPBS blank.
• The conversion from O.D. to mg/mL is (O.D. X Dilution Factor)/1.37