Affinity Media

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Overview

Ready-to-use, activated, and protein A media are available for affinity purification of biomolecules.

	Matrix	Functional Group	Specificity	Capacity	Working pH	Pressure Limit	Applications
Ready-to-Use Affinity Media
UNOsphere SUPrA	Highly cross-linked polyacrylamide polymer	Recombinant protein A	Immunoglobulins/ Antibodies	25 to 30 mg/ml	3–11	100–600 cm/hr	Monoclonal Antibody purification
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 protiens; 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		2–10	15 psi (1 bar)	Purification of IgG from ascites, serum, and culture fluid; with MAPS buffer system, purification of 10 mg mouse IgG₁ per ml of gel is possible
Affi-Prep protein A	Pressure stable polymer	Protein A 2 mg/ml	IgG		2–10	1,000 psi (70 bar)	Purifies 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 proteins	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 proteins	0.17–0.5 ml serum/ml Gel	2–11	15 psi (1 bar)	Produes 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;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 ≥10 µmol/ml	-NH₂	35 mg/ml	3–11	15 psi (1 bar)	For coupling proteins with pl 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 pl <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	2–11	15 psi (1 bar)	Carbodiimide coupling of carboxyl-containing ligand

* Refer to Bulletin 3193 for purification conditions.

Purification of a Monoclonal Antibody

Profinity IMAC Resin

UNOsphere SUPrA Media

Profinity eXact Resin

Profinity eXact purification resin chemical compatibility.*
Lysis solutions	Bacterial lysis and extraction reagent (Bio-Rad) B-PER protein extraction reagent in phosphate buffer (Pierce Biotechnology, Inc.) B-PER protein extraction reagant in Tris buffer* (Pierce) BugBuster protein extraction reagent (Novagen, Inc.) FastBreak cell lysis reagent (Promega Corporation)
Protease inhibitors	1x protease inhibitor cocktail (BD Biosciences Pharmingen) 2x protease inhibitor cocktail set 1 (Merck UK: Calbiochem) Complete protease inhibitor cocktail tablets (Roche) 0.5 mM PMSF 0.1 mM TLCK 0.1 mM TPCK
Detergents	5% (v/v) Triton X-100 5% (v/v) NP-40 5% (v/v) Tween 20 5% (w/v) octylthioglucoside 5% (w/v) n-dodecyl b-D-maltoside 5% (w/v) CHAPS 5% (w/v) CHAPSO
Reducing agents	20 mM β-mercaptoethanol 10 mM DTT 5 mM TCEP
Chelating reagents	20 mM EDTA 20 mM EGTA
Buffer reagents	50 mM Tris-acetate, pH 7.2 50 mM Tris-phosphate, pH 7.2 50 mM HEPES, pH 7.2 50 mM PIPES, pH 7.2 50 mM MOPS, pH 7.2 50 mM MES, pH 7.2
Additives	20% (v/v) glycerol 20% (v/v) ethylene glycol 20% (v/v) ethanol 20% (w/v) sorbitol 20% (w/v) sucrose 200 mM imidazole 200 mM sodium acetate 100 mM sodium borate 100 mM sodium citrate 100 mM sodium sulfate 15% (w/v) ammonium sulfate 5% (v/v) DMSO 5 mM MgCl₂ 5 mM CaCl₂

* Compatibility determined using Profinity eXact control lysate; some reagents, like ammonium sulfate, are protein-dependent.
** Chloride ions trigger cleavage of target proteins.

Boronate Applications

Category Products

Protein A Media
Bio-Rad offers protein A media for different mAb purification needs. UNOsphere SUPrA is designed for process scale purification workflows. Affi-Gel and Affi-Prep protein A media yield highly purified ...More »

Profinity IMAC Resins
Profinity IMAC resin is an affinity chromatography support for the purification of recombinant histidine (His)-tagged proteins.

Profinity eXact Purification Resin
The Profinity eXact purification resin consists of a highly engineered subtilisin protease conjugated to an agarose-based matrix.

Activated Affinity Media
Activated media for spontaneous ligand immobilization allow for the creation of an affinity matrix of choice by coupling a ligand, such as an antibody, enzyme, or antigen.

Ready-to-Use Affinity Media
Base matrices are available with ligands already coupled for convenience. Each ligand has unique specificity and can be used to capture specific molecules.

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Composition	Highly cross-linked polymer
Particle Size Range	53–61 µm
Ligand	Recombinant Protein A
Coupling Chemistry	Epoxy
Working pH range	3–11
Dynamic Binding Capacity*	30 ± 3 mg/ml at 150 cm/hr 25 ± 2 mg/ml at 300 cm/hr 20 ± 2 mg/ml 450 cm/hr (Minimum spec: 20 mg/ml at 300 cm/hr)
Chemical Stability	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)
Cleaning-In-Place (CIP) Solutions	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

Application	Application Buffer	Molecules in V_o	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

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Affinity Media

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