Activated Supports

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Overview

Activated media for ligand immobilization allow the researcher to create an affinity matrix of choice by coupling a ligand such as an antibody, enzyme, antigen, or receptor. There are multiple coupling methods. Three methods will be discussed here.

Related Topics: Affinity Chromatography, Affinity Purification of Tagged Recombinant Proteins, and Protein A/G Affinity.

Cyanogen Bromide Activation

Cyanogen bromide activation is the most common method for preparing affinity gels. It reacts with the hydroxyl groups on agarose supports to form cyanate esters and imidocarbonates. These groups react with primary amines to couple the protein onto the agarose matrix. Because cyanate esters are more reactive than are cyclic imidocarbonates, the amine will react mostly with the ester, yielding isourea derivatives, and partially with the less reactive imidocarbonate, yielding substituted imidocarbonates.

Disadvantages include the toxicity of cyanogen bromide and its sensitivity to oxidation. Cyanogen bromide activation also involves the attachment via an isourea bond, which is positively charged at neutral pH and thus unstable. Consequently, isourea derivatives may act as weak anion exchangers.

Cyanogen bromide activation

Epoxide Activation

Ligands containing amino, thiol, or hydroxyl groups can be coupled to the epoxide through an epoxy ring-opening reaction under mild conditions. Following immobilization, active groups remaining on the resin need to be deactivated or blocked to avoid undesirable reaction with proteins of interest during affinity chromatography. They can be deactivated or blocked by mixing the resin with 1 M ethanolamine, pH 8–l9.

Carbodiimide Activation

This reaction mechanism involves two steps: the activation of carboxyl groups with displacement by the nucleophile, R-NH2, thereby releasing the EDAC as a soluble urea derivative. The carboxyl groups can be present on the ligand or on the matrix. The activated carboxyl groups react with the amino groups in the reaction mixture. The illustration shows the EDAC coupling reaction for a ligand with a terminal carboxyl group to Bio-Rad’s Affi-Gel® 102 gel.

Bio-Rad's activated affinity chromatography media.

 
Matrix
Functional
Group
Specificity Capacity Working pH Pressure
Limit
Applications
Activated Media for Spontaneous Ligand Immobilization
Profinity™ epoxide Pressure-stable polymer based on UNOsphere beads Epoxy group Nucleophiles,
amino, thiol,
COOH
36–40 mg/ml IgG 1–14 Up to 80 psi (5.5 bar) Activated matrix for the immobilization of various ligands (for example, protein A,
StrepTactin, and immunoglobulins)
Affi-Gel 10 Crosslinked agarose

N-hydroxy-
succinimide µmol/ml
≥10 µmol/ml

–NH2 35 mg/ml 3–11 15 psi (1 bar) For coupling proteins with pI 6.5–11
Affi-Gel 15 Crosslinked agarose N-hydroxy-
succinimide µmol/ml
≥9 µmol/ml
–NH2 35 mg/ml 3–11 15 psi (1 bar) For coupling proteins with pI <6.5
Affi-Gel Hz Crosslinked agarose Hydrazide Oxidized carbohydrates 1–5 mg/ml 2–10 15 psi (1 bar) Immobilization of immunoglobulins
and other glycoproteins via
carbohydrate moieties
Affinity Media Using Carbodiimide Activation
Affi-Gel 102 Crosslinked agarose –NH2
16 ± 4 meq/ml
–COOH 40 mg/ml 2–11 15 psi (1 bar) Carbodiimide coupling of carboxyl-containing ligand

Ready-to-Use Activated Media

There are a number of preactivated media that include dyes, metals, and other ligands. The correct choice is dictated by the group available in the ligand molecule and the nature of the binding interaction with the molecule to be purified. High selectivity and capacity make this technique ideally suited to the isolation of specific components from complex biological mixtures.

Bio-Rad's ready-to-use activated affinity chromatography media.

 
Matrix
Functional
Group
Specificity Capacity Working pH Pressure
Limit
Applications
Ready-to-use Affinity Media
Affi-Gel protein A Crosslinked agarose Protein A
2 mg/ml
IgG 6–15 2–10 15 psi
(1 bar)
Purification of IgG from ascites, serum, and culture fluid; with MAPS™ buffer system; purification for 10 mg mouse IgG, per ml of media is possible
Affi-Prep® protein A Pressure-stable polymer Protein A
2 mg/ml
IgG 7–12 2–10 1,000 psi (70 bar) Purifies IgG from ascites, serum, and culture fluid; pressure-stable media for process-scale applications
Affi-Gel Blue Crosslinked agarose Cibacron Blue F3GA 1.9 mg/ml Albumin, general ≥11 mg/ml 2–10 15 psi
(1 bar)
Binds many nucleotide-requiring enzymes, albumin, and other proteins
DEAE Affi-Gel Blue Crosslinked agarose Cibacron Blue F3GA and DEAE Albumin and serum proteins 0.14 ml serum/ml 2–10 15 psi
(1 bar)
Purifies protease-free IgG from ascites, serum, and culture fluid with minimal sample preparation
CM Affi-Gel Blue Crosslinked agarose Cibacron Blue F3GA and CM Albumin and serum proteins 0.17–0.5 ml serum/ml 2–11 15 psi
(1 bar)
Produces albumin and protease-free antibody preparation from serum without prior dialysis
Affi-Prep polymyxin Pressure-stable polymer Polymyxin
2–4 mg/ml
Endotoxins >5 mg/ml 2–10 1,000 psi (70 bar) Endotoxin removal
Affi-Gel boronate Polyacrylamide gel Boronate 1.05 ± 0.15 meq/g cis-diols 130 µmol
sorbitol/ml
2–10 15 psi
(1 bar)
Adsorption of cis-hydroxyl-containing molecules, including sugars, nucleotides, and glycopeptides
Profinity™ IMAC Pressure-stable polymer based on UNOsphere beads IDA, provided charged with Ni2 and uncharged Histidine ≥15 mg/ml* 2–10 100 psi
(6.8 bar)
Purification of recombinant proteins tagged with His; can be charged with other transition metals
Profinity GST** Pressure-stable polymer based on UNOsphere beads Immobilized glutathione GST-tagged proteins ≥10 mg/ml 2–10 45 psi
(3.1 bar)
Purification of recombinant GST-tagged proteins
Profinity eXact™*** Crosslined 6% agarose Subtilisin protease Subtilisin prodomain ≥3 mg/ml tag-free protein 2–10 10 psi
(minus system pressure)
Generation of native, tag-free protein by on-column purification and cleavage
UNOsphere SUPrA™ Highly cross-linked polymer Recombinant protein A Immunoglobulins/
antibodies
30 ±
3 mg/ml
3–11 100–600 cm/hr Purification of IgG at lab and process scale

* Refer to bulletin 3193 for purification conditions.
** For example, Profinity GST resin is available only in prepacked cartridges.
***Profinity eXact purification resin.

Related Content

Literature
Number Description Download
5712 The Profinia Protein Purification System Simplifies Antibody Purification With Protein A, Rev A Click to download
5925 Affinity Purification Buffer Kit Product Information Sheet, Rev B Click to download
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