Introduction

Purification protocols for immunoglobulins generally include affinity binding to Protein A or G chromatography media, washing unbound proteins, and eluting with a low pH buffer. The resulting purified antibodies are often too dilute for downstream studies or long-term storage. Dialysis is often necessary to concentrate the antibody and/or exchange the buffer into one that preserves protein activity. Ultrafiltration achieves the same result faster, with up to 99% immunoglobulin recovery and one-step salt removal (see Concentration, Desalting, and Buffer Exchange protocol).

Method and Results

Purification and concentration of Rabbit IgG with Prosep® A and Prosep® G Montage® Antibody Purification Kits and Amicon® Ultra-15 mL centrifugal filters.

A. Purification of IgG using Montage® Kits

  1. Equilibrate the Prosep® A or Prosep® G resins with 10 mL of binding buffer and spin for 5 min at 500 xg
  2. Pre-clarify the serum sample by passing through a 0.22 µm Steriflip® GP filter.
  3. Dilute the sample 1:1 with binding buffer A and centrifuge the device at 100-150 x g for 20 min.
  4. Wash the spin column with 20 mL of binding buffer A to remove unbound contaminants by centrifuging the column for 5 min at 500 x g.
  5. Elute the samples by adding 10 mL of elution buffer B2 into a fresh centrifuge tube containing 1.3 mL of neutralization buffer.
  6. Measure the total protein concentration of the sample using an appropriate concentration method, such as UV absorbance at 280 nm.

B. Concentration of IgG using Amicon® Ultra-15 30kDa MWCO devices

  1. Add the neutralized eluted fraction into Amicon® Ultra 15 mL 30 kDa MWCO device.
  2. Centrifuge for 10 to 20 min at 4000 x g in a swinging bucket rotor or 5000 x g in a fixed angle rotor until the sample has reached the desired concentration.
Concentration of rabbit IgG with Amicon Ultra-15 devices. The IgGs were purified using Montage PRSEP-A pr PROSEP-G Antibody Purification Kits. The lines show IgG volume reduction, while the bars show a proportional increase in IgG concentration after 20 min of centrifugation time.

Figure 1.Concentration of rabbit IgG with Amicon Ultra-15 devices. The IgGs were purified using Montage PRSEP-A pr PROSEP-G Antibody Purification Kits. The lines show IgG volume reduction, while the bars show a proportional increase in IgG concentration after 20 min of centrifugation time.

SD-PAGE gel of purified rabbit IgG before and after concentration with Amicon Ultra-15 devices.

Figure 2.SD-PAGE gel of purified rabbit IgG before and after concentration with Amicon Ultra-15 devices.

Materials
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