Protocols

Antibody Purification Protocol

Posted on Monday, May 03, 2004

Description
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

Procedure
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

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Submitted by: manny440