Affi-Gel Protein A Media

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Overview

Protein A Capacity Table
Capacities of Protein A Supports
Immunoglobulin Affi-Gel Protein A
Capacity (mg/ml)		 Affi-Prep Protein A
Capacity (mg/ml)
Mouse lgG1 6–10 7–9
Mouse lgG2a 6–10 7–9
Mouse lgG2b 6–10 7–9
Mouse lgG3 6–10 7–9
Mouse lgM* 6–10 7–9
Human lgG 15 10–12
Sheep lgG 8–10 7–9
Cow lgG 8–10 7–9
Horse lgG 8–10 7–9
Goat lgG 8–10 7–9
Rabbit lgG 8–10 7–9
Dog lgG 8–10 7–9
Pig lgG 8–10 7–9

* Approximately 50% of all mouse lgMs bind using the MAPS buffer system.

Affinity Media Selection Guide
Affinity Media Selection Guide
 
  Matrix Functional Group Specificity Capacity Working pH Pressure Limit Applications
 
Ready-to-Use Affinity Media
 
Profinity IMAC Pressure-stable polymer based on UNOsphere beads IDA, provided charged with Ni2+ and uncharged Histidine ≥15 mg/ml* 1–14 100 psi (6.8 bar) Purification of recombinant His-tagged proteins; can be charged with other transition metals
Profinity GST Pressure-stable polymer based on UNOsphere beads Immobilized glutathione GST-tagged proteins ≥10 mg/ml 1–14 45 psi (3.1 bar) Purification of recombinant GST-tagged proteins
Profinity eXact*** Crosslinked 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
Affi-Gel protein A Crosslinked agarose Protein A 2 mg/ml IgG See page 53 2–10 15 psi (1 bar) Purification of IgG from ascites, serum, and culture fluid; with MAPS buffer system, purification of 10 mg mouse IgG, per ml of gel is possible
Affi-Prep protein A Pressure-stable polymer Protein A 2 mg/ml IgG See page 53 2–10 1,000 psi (70 bar) Purified IgG from ascites, serum, and culture fluid; pressure-stable support 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 protiens 0.14 ml serum/ml gel 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 protiens 0.17–0.5 ml serum/ml gel 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 cis-diols

meq/g		 130 µmol sorbitol/ml 2–10 15 psi (1 bar) Adsorption of cis-hydroxyl-containing molecules, including sugars, nucleotides, and glycopeptides
               
 
Activated Media for Spontaneous Ligand Immobilization
 
Profinity epoxide Pressure-stable polymer based on UNOsphere beads Epoxy group Nucleophiles; amini, thiol, -COOH 36–40 mg/ml lgG 1–14 Up to 80 psi (5.5 bar) Activated matrix for the immobilization of barious ligands (for example, protein A, StrepTactin, and immunoglobulins)
Affi-Gel 10 Crosslinked agarose N-hydroxy-succinimide ≥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 ≥9 µmol/ml -NH2 35 mg/ml 3–11 15 psi (1 bar) For coupling proteins with pI 6.5–11
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 molecules
               
 
Affinity Media Using Carbodiimide Activation
 
Affi-Gel 102 Crosslinked agarose -NH2 16 ± 4 meq/ml -COOH 40 mg 2–11 15 psi (1 bar) Carbodimide coupling of carboxyl-containing ligand
 
* Refer to bulletin 3193 for purification conditions.
** For example, Profinity GST resin is only available in prepacked cartridges. Refer to page 63.
***Profinity eXact purification resin, see page 97 for more information.
UNOsphere SUPrA Technical Properties
Technical Properties of UNOsphere SUPrA Affinity Media
Composition Highly Crosslinked Polyacrylamide Polymer
Particle Size Range 53–61 µm
Ligand Recombinant Protein A
Coupling Chemistry Epoxy
Dynamic Binding Capacity1 150 cm/hour, 30±3 mg/ml
300 cm/hour, 25±2 mg/ml
450 cm/hour, 20±2 mg/ml
Chemical Stability2 10 mM Hydrochloric Acid
6 M Guanidine Hydrochloride
0.1 M Arginine (pH 2.8)
0.1 M Citrate (pH 2.8)
0.1 M Glycine (pH 2.8)
Working pH Range 3–11
Cleaning-In-Place (CIP) 6 M Guanidine Hydrochloride
10 mM Hydrochloric Acid
0.1 M Sodium Hydroxide
1 M Acetic Acid/20% Ethanol
Recommended Mobile Phase Velocity Range 100–600 cm/hr
Temperature Stability 4–40°C
Delivery Conditions 50% slurry in 20% Ethanol
Storage Conditions 4–8°C

1 - Minimum 20 mg/ml at 300 cm/hr; 10% breakthrough capacity determined with 1.0 mg/ml polyclonal human IgG in 1.1 x 10 cm column
2 - No significant change in chromatographic performance after 24 hour storage at room temperature.

Boronate Applications
Boronate Applications
Application Application Buffer Molecules in Vo Retained Molecules Elution Buffer Support Used
Adenylate cyclase assay HEPES pH 7.5 + MgCl2 cAMP ATP, AMP, and adenosine 0.05 M NaOAc Bio-Gel P-150 boronate gel
Isolation of catecholamines from urine 0.1 M phosphate pH 7.0 + EDTA Other urine components and DOPA Norepinephrine, epinephrine 0.025 N HCI Boric acid gel (Sigma-Aldrich)
Modified nucleosides in urine 0.25 M NH4OAc, pH 8.8 Thymidine, adenine Pseudouridine 0.1 M HCOOH Bio-Gel P-2 gel, 200–400 mesh, boronate
Separation of mono- and oligonucleotides Triethyl ammonium Deoxyribonucleotides Ribonucleotides H2O and others Dihydroxyboryl methacrylate
Sugars 0.05 M N-methyl-morpholinium-Cl, pH 7.5 + 1 M NaOAc Erythritol, adonitol, sucrose, and D-glucose L-arabinitol, xylitol, D-mannitol, dulcitol, sorbitol, and D-fructose* Isocratic run, elution buffer same as application buffer Dihydroxyboryl cellulose
Assay of ribonucleotide reductase Tris, MgCl2 dUMP, dCMP CDP Citrate Dihydroxyboryl Cellulose Dowex 1(AG) resin

* In order of increasing retention by support.

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Structure
Structure

Affi-Gel boronate-derivatized polyacrylamide gel has affinity for coplanar adjacent cis-hydroxyl groups (cis-diols) and a high binding capacity, which provides highly efficient separation of low molecular weight molecules, such as nucleotides, nucleosides, catecholamines, and sugars. It has a sorbitol capacity of 130 µmol/ml.



Chromatography on Affi-Gel protein A media yields highly purified immunoglobulins (IgGs), selectively removes IgG prior to analysis of other immunoglobulin classes, or adsorbs immune complexes for antigen purification. Protein A binds to the Fc region of immunoglobulins, especially IgG from mammalian species.

Advantages include:

  • High purity of IgGs
  • High affinity for mammalian IgG
  • High capacities for mouse IgG1 as well as other subclasses with MAPS optimized buffer

With Affi-Gel protein A media and MAPS II buffers, you can purify up to 10 mg of IgG1/ml of gel. This is 8–10 times higher than is possible with standard methods. In addition, the MAPS process and Affi-Gel protein A media permit greater binding of mouse IgG1 than do immobilized protein G media.

Affi-Gel Protein A Support

153-6153
5 ml, ready-to-use protein A crosslinked agarose support

List Price:   $300.00
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Affi-Gel Protein A Support

153-6154
50 ml, ready-to-use protein A crosslinked agarose support

List Price:   $2,195.00
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Affi-Gel Protein A MAPS II Kit

153-6159
Ready-to-use affinity media kit, includes 5 ml Affi-Gel protein A support, Affi-Gel Protein A MAPS II buffers, 1 x 10 cm Econo-Column column; for 500 mg of mouse IgG1

List Price:   $385.00
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Hazardous  
Protein A MAPS II Binding Buffer

153-6161
Optimized buffer concentrate, for use with Affi-Gel and Affi-Prep protein A media, makes 5 L

List Price:   $765.00
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Number Description Options
5334 A Method for Rapid, Large-Scale Removal of Albumin and IgG From Human Serum Using the BioLogic DuoFlow Chromatography System, Rev A Click to download
LIT88 Instruction Manual, Affi-Gel Protein A MAPS II Kit Click to download [ Add to Cart ]
LIT153 Instruction Manual, Econo-Pac Protein A Kit, Protein A Columns, Rev B Click to download [ Add to Cart ]