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Size Exclusion Chromatography Affinity Chromatography Enzyme & Antibody Immobilization


AGAROSE BEAD TECHNOLOGIES No part of this book may be reproduced or transferred in any form or by any means, graphic, electronic, or mechanical, including photocopying, recording, taping, or by any information storage retrieval system, without the written permission of the author.

Š 2011. AGAROSE BEAD TECHNOLOGIES All rights reserved.

All products for research use. Not use in diagnostic or therapeutic procedures. Preparation and purification of recombinant proteins/peptides containing neighbouring His residues may require a licence under US pat 5,284,933 and US pat. 5,310,663, including corresponding patents (assignee: Hoffman La Roche.Inc).

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GST PURIFICATION

SIZE EXCLUSION CHROMATOGRAPHY LOW PRESSURE • Plain & Crosslinked agarose beads HIGH PRESSURE • Rapid Run™ agarose beads

7 7 10 10

LOW PRESSURE • Chelating agarose beads -

12 14 14

Test Kit Bulk Resins Pre-Packed Columns Technical Performance Data

HIGH PRESSURE • Chelating Rapid Run™ agarose beads

21 21

24

LOW PRESSURE • Protein A agarose beads

24 26

- Test Kit - Bulk Resins - Pre-Packed Columns

- Cartridges

ACIDIC GROUPS LOW PRESSURE • Aminoethyl agarose beads

34 34 34

- Test Kit - Bulk Resins

34 36

- Bulk Resins

AMINO GROUPS LOW PRESSURE • Glyoxal agarose beads

ANTIBODY PURIFICATION

HIGH PRESSURE

29

- Bulk Resins - Cartridges - Technical Performance Data

HIGH PRESSURE • Aminoethyl Rapid RunTM agarose beads

- Cartridges - Bulk Resins

• Protein A Rapid Run™ agarose beads

29

ENZYME & ANTIBODY IMMOBILIZATION

AFFINITY CHROMATOGRAPHY HIS-TAG PURIFICATION

29

LOW PRESSURE • Glutathione agarose beads

37 37 37

- Test Kit - Bulk Resins - Technical Performance Data

HIGH PRESSURE • Glyoxal Rapid RunTM agarose beads

39 39

- Bulk Resins

27 27

ACCESSORIES EMPTY COLUMNS

40

EMPTY CARTRIDGES

42 -3-


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Agarose Bead Technologies (ABT) is dedicated to the research and development of biotechnology products derived from agarose. The company offers a wide range of activated agarose beads for affinity chromatography and enzyme and antibody immobilization processes, as well as a variety of non activated agarose beads for size exclusion chromatography. Agarose Bead Technologies relies on a strong Quality Program of selecting the finest agarose available on the market to manufacture a growing list of beaded products. With several years experience in this technically demanding field, ABT is expanding its worldwide presence with distribution partners throughout the globe and has sales offices in the US, the UK and Spain. It continues to produce new products annually such as our high flow rate/pressure Rapid Run™ beads to meet our customer’s unique requirements. ABT encourages the development of customized beaded products in cooperative relationships with its clients. ABT’s mission is to focus its efforts in producing highly specialized products such as agarose beads having the widest range within its manufacturing lines, thus positioning itself as a specialist in this growing market segment.

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SIZE EXCLUSION CHROMATOGRAPHY Low Pressure PLAIN & CROSSLINKED AGAROSE BEADS Agarose is a very inert polysaccharide which forms hydrophilic and high gel strength gels at low concentrations. Agarose beads are microspheres of agarose gels with different particle diameters and concentrations. Small spherical particles of agarose act as a porous gel to filter or separate a mixture of molecules according to their individual sizes. Due to the composition (easy to activate), the agarose beads may be prepared to bind biomolecules in a reversible or irreversible manner.

Plain and crosslinked agarose beads are used in Gel Filtration Chromatography (or Molecular Exclusion Chromatography) as well as for activating beads for biomolecule purification or immobilization. Plain and crosslinked agarose beads can also be used for activating processes, internal surface and to its composition (inert polysaccharide), agarose is an ideal medium for the preparation of activated beads. ABT offers a wide range of plain and crosslinked agarose beads with different agarose concentrations (2, 4, 6, 8 & 10%) in different particle size distributions Macro (150-350 µm), Standard (50-150 µm) and Fine (20-50 µm). • The widest range of different agarose concentrations.

• Excellent chemical and physical stability.

• Different pore sizes.

• Negligible non specific adsorption.

• Broad fractionation range.

• For batch or column procedures.

• Crosslinked (autoclavable).

• Three different particle sizes.

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-8-


LOW PRESSURE SIZE EXCLUSION CHROMATOGRAPHY

PLAIN & CROSSLINKED AGAROSE BEADS

TECHNICAL SPECIFICATIONS

PLAIN AGAROSE BEADS

CROSSLINKED AGAROSE BEADS

Spherical

Spherical

BEAD GEOMETRY CROSSLINKED

No

CHEMICAL STABILITY

Yes

Stable in strong acid and basic solutions

MECHANICAL STABILITY

(1)

Stable in very strong acid and basic solutions also in dissociating reagents(1) Suitable and comparable to comercial counterparts

BEAD AGAROSE %

2%

4%

6%

8%

ANTIMICROBIAL AGENT

10%

2%

4%

6%

8%

10%

20% Ethanol

RECOMMENDED STORAGE TEMPERATURE

2 - 25º C

2 - 25º C

(2)

(2)

(2)

(2)

(2)

(2)

(2)

(2)

X: Product Quantity. Standard (500 ml, 1 L or 10 L). Fine (250 ml, 500 ml, 1L). Larger amounts available on request. * MACRO: ~150 -350µm; ~STANDARD: ~50-150µm; FINE 20-50µm. 1 See stability table in Plain & Crosslinked Agarose Beads Procedure for Use. 2 Customized product. Ask for information.

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High Pressure RAPID RUN™ AGAROSE BEADS

ABT has developed Rapid RunTM high throughput beads to meet a media market demand for industrial process separations. Their rigidity and mechanical resistance permits high flow rates, with good resolution in a minimum time, making these beads ideal for process-scale use. Rapid RunTM beads are based on highly crosslinked 4% and 6% agarose matrices, respectively, which give excellent physical and chromatographic qualities. Rapid RunTM beads are an ideal support for the immobilization of ligands for Affinity Chromatography and base media support for producing IEX and Hydrophobic interaction chromatography resins. These media are the accepted standard for laboratory as well as large scale applications.

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Rapid RunTM beads exhibit the following main characteristics: • High mechanical resistance.

• Good binding capacity.

• High flow/pressure properties.

• Low non specific adsorption.

• High physical and chemical stability. • Thermally stable. • Scalable.

• Good reproducibility.


HIGH PRESSURE SIZE EXCLUSION CHROMATOGRAPHY

TECHNICAL SPECIFICATIONS

4% RAPID RUNTM AGAROSE BEAD STANDARD

6% RAPID RUNTM AGAROSE BEAD STANDARD

BEAD GEOMETRY

Spherical

BEAD STRUCTURE

Highly cross-linked agarose ~ 50 – 150 µm

BEAD SIZE RANGE TEMPERATURE STABILITY

Autoclavable at 120 ºC for 20 min in H20

pH STABILITY CHEMICAL STABILITY BEAD AGAROSE %

pH 1.8-14 short term / pH 3.8-13 long term Most commonly used aqueous and organic solutions including: 1 M NaOH, 8 M Urea, 5 M guanidine HCl, 75% ethanol 4%

6%

4RRS-X

6RRS-X

~ 3 X 107

~ 4 X 106

MAXIMUM FLOW RATE AT 15 cm BED HEIGHT1

≥ 500 cm /h

≥ 1000 cm /h

MAXIMUM PRESSURE AT 15 cm BED HEIGHT1

≥ 150 kPa

≥ 300 kPa

CAT. No. EXCLUSION LIMIT

ANTIMICROBIAL AGENT RECOMMENDED STORAGE TEMPERATURE

20% Ethanol 2 - 30º C

1 Column; XK 16/40. System: Akta Purifier UPC 100. Maximum flow rates: The highest flow that beads withstood for 1 minute without collapsing and the pressure reaching 1MPa. X: Product Quantity (500 ml, 1 L or 10 L).

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AFFINITY CHROMATOGRAPHY HIS-TAG PURIFICATION Purification of proteins is a vital part of modern research. Impure extracts generally contain a wide range of proteins with diverse biological functions and different chemistry, which need to be separated. Affinity Chromatography (IMAC) is the purification technique which is widely used. It is based on the interaction between certain superficial protein residues (histidines, cysteines and to a lesser extent tryptophans), with transition metal cations, forming chelates. These transition metals are bound through a chemical reaction to the agarose beads, giving the agarose an activation degree. ABT manufactures two types of chelating beads utilizing standard crosslinked beads and the new highly crosslinked Rapid RunTM beads.

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The binding between the metal and the agarose bead is by means of the IDA. IDA crosslinked agarose resin consists of iminodiacetic acid groups ligated by stable ether linkages via a spacer arm. IDA is a tridentate chelating agent, covalently coupled to crosslinked agarose beads. This resin is loaded with a divalent metal (Ni2+, Cu2+, Zn2+ or Co2+). The resulting resin (ready to use) is ideal for rapid purifications of His-tagged proteins. In comparison with other chelating resins such as NTA-agarose, the IDA has three sites available for the interaction with metal ions, instead of the four with NTA. IDA resins are usually more easily regenerated allowing a better elution of the fused proteins bound with smaller concentrations of imidazole.


HIS-TAG PURIFICATION AFFINITY CHROMATOGRAPHY

The product range covers 4 different types of metal and two different densities of groups on the beads. The choice of resin depends on the objectives/priorities for each purification (binding capacity/selectivity) and the type of protein to be purified (easy or difficult to separate). The easiest strategy is to screen with the correct Test Kit, and decide on options based on both the protein and the purpose and objectives of the purification. The user can optimize the best recovery system by using the Test Kit approach. Once the user has chosen the best option, ABT offers further different product formats: • Bulk Resins

• Pre-Packed Columns

• Cartridges

Nickel & Cobalt are the most commonly used metal ion for IMAC purifications. Nickel/ Cobalt Rapid RunTM beads combine the advantages of the metal with the high flow rates of the Rapid RunTM resin. This product is excellent for large scale His-tagged protein purifications.

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Low Pressure CHELATING AGAROSE BEADS

Test Kit Screening The choice of the best product depends on the objectives /priorities for each purification (binding capacity /selectivity) and the type of protein to be purified (easy or difficult to separate). The easiest strategy is to screen with the correct Test Kit, and decide on options. • Most suitable Test Kit for range finding. • All protein type solutions. • Selection of the most advisable product.

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HIS-TAG PURIFICATION AFFINITY CHROMATOGRAPHY

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Low Pressure CHELATING AGAROSE BEADS

Bulk Resins ABT offers resins for purifications of histidinetagged proteins by Immobilized Metal Affinity Chromatography (IMAC). • Different grades of activation to optimize the relationship between binding capacity and purification selectivity. • Resins charged with Ni, Cu, Zn or Co as well as metal free. • For batch or column purifications. • Different quantities (25, 100 & 500 ml). Larger amounts available on request.

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(25, 100 or 500 ml)


HIS-TAG PURIFICATION AFFINITY CHROMATOGRAPHY

Pre-Packed Columns

TECHNICAL SPECIFICATIONS COLUMN MATERIAL

Polypropylene column and Polyethylene frit

BED VOLUME

1 ml / 5 ml (XL)

ABT offers pre-packed ready to use columns for purifications of histidine-tagged proteins by Immobilized Metal Affinity Chromatography (IMAC). Fast and simple purification.

CROSSLINKED

Yes

• For gravity flow.

PRODUCT NAME

• Cross contamination-free. • No need of purification systems. • For native and denaturing conditions. • Available for Ni, Zn, Co and Cu chelating resins. • Contains 1 or 5 ml of gel.

BEAD AGAROSE % ANTIMICROBIAL AGENT

6% 20% Ethanol

STORAGE TEMPERATURE

2 - 8º C

PACK SIZE

CAT. No.

HIS-COLUMN HIGH DENSITY NICKEL

8 x 1 ml

6BCL-QHNi-C8

HIS-COLUMN LOW DENSITY NICKEL

8 x 1 ml

6BCL-QLNi-C8

HIS-COLUMN HIGH DENSITY ZINC

8 x 1 ml

6BCL-QHZn-C8

HIS-COLUMN LOW DENSITY ZINC

8 x 1 ml

6BCL-QLZn-C8

HIS-COLUMN HIGH DENSITY COBALT

8 x 1 ml

6BCL-QHCo-C8

HIS-COLUMN LOW DENSITY COPPER

8 x 1 ml

6BCL-QLCu-C8

HIS-XL COLUMN HIGH DENSITY NICKEL

5 x 5 ml

6BCL-QHNi-C5

HIS-XL COLUMN LOW DENSITY NICKEL

5 x 5 ml

6BCL-QLNi-C5

HIS-XL COLUMN HIGH DENSITY ZINC

5 x 5 ml

6BCL-QHZn-C5

HIS-XL COLUMN LOW DENSITY ZINC

5 x 5 ml

6BCL-QLZn-C5

HIS-XL COLUMN HIGH DENSITY COBALT

5 x 5 ml

6BCL-QHCo-C5

HIS-XL COLUMN LOW DENSITY COPPER

5 x 5 ml

6BCL-QLCu-C5 - 17 -


CHELATING AGAROSE BEADS TECHNICAL PERFORMANCE DATA

- 18 -


HIS-TAG PURIFICATION AFFINITY CHROMATOGRAPHY

- 19 -


High Pressure CHELATING RAPID RUN TM AGAROSE BEADS Cartridges

TECHNICAL SPECIFICATIONS

Nickel Rapid RunTM Cartridges (1ml) & Nickel Affinity Cartridges (5ml) are used for purification of histidine-tagged proteins in native or denaturing conditions.

PRODUCT NAME

NICKEL RAPID RUNTM Cartridges 1ml

BEAD (Geometry, size)

Spherical, Fine: ~ 20 - 50 µm

CROSS-LINKED

Highly crosslinked 6% agarose

• Easy changeover: no need for optimization or protocol change. • Great adaptability: Cartridges suitable for MPLC, FPLCTM, ÄKTA designTM, batch, gravity, peristaltic pump & syringe. • High purity achieved in one purification step, comparable to market standards.

LIGAND MAX. BACK PRESSURE MATRIX

Stable in all commonly used reagents (1)

RECOMENDED FLOW RATE

1 ml / min

MAXIMUM FLOW RATE

4 ml / min

APPLICATION

Automated liquid chromatography (MPLC, FPLCTM, ÄKTA designTM) peristaltic pump & syringe

CAT. No. DESCRIPTION CARTRIDGE PORTS ANTIMICROBIAL AGENT STORAGE TEMPERATURE

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IDA 0.3 MPa, 3 bar

1 See stability table in Procedure for Use. X: Quantity of cartridges (2 or 5). ABT offers the option to prepack empty cartridges. See page 43.

6RRFNi-Ctg1-X X cartridges 1ml resin each one Inlet port & outlet port: 10 – 32 (1/16´´) female 20% Ethanol 2 - 8º C


HIS-TAG PURIFICATION AFFINITY CHROMATOGRAPHY

TECHNICAL SPECIFICATIONS PRODUCT NAME

NICKEL Affinity Cartridges 5ml

BEAD (Geometry, size)

Spherical, Fine: ~ 20 - 50 µm

CROSSLINKED

Highly cross-linked 6% agarose

LIGAND

IDA

MATRIX

Stable in all commonly used reagents (1)

RECOMMENDED FLOW RATE APPLICATION CAT. No. DESCRIPTION CARTRIDGE PORTS ANTIMICROBIAL AGENT STORAGE TEMPERATURE

1

5 ml /min Automated liquid chromatography (MPLC, FPLCTM, ÄKTA designTM) peristaltic pump & syringe AF6Ni-Ctg5-X X cartridges 5 ml resin each one Standard 10 – 32 fitings without aditional connectors 20% Ethanol 2 - 8º C

See stability table in Procedure for Use. X: Quantity of cartridges (1 or 5). Larger amounts available on request. Note: Binding capacity was tested using purified Dehydroxyacetone (6 x His) and the result was 110 mg DHAK-(6x His) purified / ml medium. This is only an indicative value because binding capacity can be affected by several factors such as sample concentration, binding buffer and the flow rate during sample application. .

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HIS-TAG PURIFICATION AFFINITY CHROMATOGRAPHY

High Pressure CHELATING RAPID RUN TM AGAROSE BEADS Bulk Resins Nickel & Cobalt are the most commonly used metal ions for IMAC purifications. Nickel/ Cobalt Rapid RunTM beads combine the advantages of the metal with the high flow rates of the Rapid RunTM resin. This product is excellent for large scale His-tagged protein purifications. Nickel/Cobalt Rapid RunTM chelating agarose beads are designed for large scale downstream purification of His-tagged proteins using IMAC technology. This resin combines the advantages of Nickel/ Cobalt with the high flow rates of the Rapid RunTM resin: these beads support 70% higher flow rates than other commercially available products.

• Easy scale up and robust function.

• Good resolution in minimal time.

• High chemical and physical stabilities. TECHNICAL SPECIFICATIONS PRODUCT NAME BEAD (Geometry, size)

NICKEL RAPID RUNTM

Spherical, Standard: ~ 50 - 150 µm

EXCLUSION LIMIT CROSSLINKED

COBALT RAPID RUNTM

~ 4 X 10 6 Highly crosslinked 6% Agarose

MAXIMUM FLOW RATE AT 15 cm bed height1

≥ 1000 cm / h

MAXIMUM PRESSURE AT 15 cm bed height1

≥ 30O kPa

CAT. No. BINDING / LOADING CAPACITY + (µmol Me2 /ml gel) LIGAND ANTIMICROBIAL AGENT STORAGE TEMPERATURE

6NiRR-X

6CoRR-X

~ 20

~ 15 Iminodiacetic acid 20% Ethanol 2 - 8º C

1 Data corresponding to the non-activated Rapid RunTM beads. Column: XK 16/40. System Äkta Purifier UPC 100. Maximum flow rate: The highest flow that beads withstood for 1 minute without collapsing and the pressure reaching 1MPa X: Product Quantity (5, 25, 100, 500 or 1.000 ml).

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AFFINITY CHROMATOGRAPHY ANTIBODY PURIFICATION PROTEIN A AGAROSE BEADS Protein A is a cell wall component of . It consists of a single polypeptide chain shaped as a cylinder, which contains five antibody–binding domains. These high affinity regions are specifically bonded to Fc region of the immunoglobulins (specially IgGs of different species). Protein A is temperature stable and it retains its native conformation even in the presence of denaturing agents. Protein A resins have been widely used to purify a wide range of immunoglobulins of different mammalian species and also to purify certain IgG subclasses that have no affinity.

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ABT offers rProtein A products with competitive advantages compared to market standards: • High IgG-binding capacity resin (around 25 mg human IgG / ml). • Milder elution condition than Protein G resin. • High stability binding of rProtein A: resin is reusable with no significant loss of binding capacity. ABT offers different product formats: • Test Kit. • Bulk Resins. • Pre-Packed Columns. • Cartridges.


ANTIBODY PURIFICATION AFFINITY CHROMATOGRAPHY

RELATIVE AFFINITY OF IMMOBILIZED PROTEIN A FOR VARIOUS SPECIES AND SUBCLASSES OF POLYCLONAL AND MONOCLONAL IgGs(1) SPECIES / SUBCLASS

PROTEIN A

PROTEIN A

Polyclonal

Monoclonal

Rabbit

++++

IgG1

++++

Cow

++

IgG2

++++

Horse

++

IgG3

____

Goat

_

IgG4

++++

Ginea Pig

++++

Sheep

+/-

+

Pig

+++

IgG2a

++++

Rat

+/-

IgG2b

+++

Mouse

++

IgG3

++

Chicken

___

Human IgG

++++

Human

Mouse IgG1

Rat

1

SPECIES / SUBCLASS

IgG1

___

Human IgM

___

IgG2a

___

Human IgD

___

IgG2b

___

Human IgA

___

IgG2c

+

Harlow, E. And Lane, D. eds. (1988). Antibodies, A. Laboratory Manual. Cold Spring Harbor Laboratory, N.Y., 617-618.

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Low Pressure PROTEIN A AGAROSE BEADS

Test Kit, Resins & Pre-Packed Columns ABT offers Protein A resins for purifications of a wide range of Immunoglobulins of different mammalian species and also to purify certain IgG subclasses that have no affinity. • Get more of your antibody: higher IgG binding capacity. • Get a better purification: higher stability binding of the rProtein A. • Save time and money: reusable. No leakage levels. • Different product formats: Test Kit, Bulk and Pre-Packed.

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TECHNICAL SPECIFICATIONS BEAD (Geometry, size)

Spherical, Standard: ~ 50-150 µm Yes

CROSSLINKED

4%

BEAD AGAROSE %

Covalent binding ( Protein A immobilized with the same orientation as CNBr activated resins) r

CHEMICAL STABILITY LIGAND DENSITY

~3 mg rProtein A / ml resin

BINDING CAPACITY

~25 mg human IgG / ml resin

ANTIMICROBIAL AGENT

20% Ethanol

STORAGE TEMPERATURE

2 - 8º C

PRODUCT

PACK SIZE

CAT. No.

PROTEIN A TEST KIT

1 x 100 µl

PA09-K-01 (Pre-Packed)

PROTEIN A AGAROSE BEADS

5 ml 25 ml 500 ml

PA09-R5 (Bulk) PA09-R25 (Bulk) PA09-R500 (Bulk)

PROTEIN A AGAROSE COLUMNS

1 x 1 ml 1 x 5 ml

PA09-C1-1 (Pre-Packed) PA09-C5-1 (Pre-Packed)


ANTIBODY PURIFICATION AFFINITY CHROMATOGRAPHY

High Pressure PROTEIN A RAPID RUNTM AGAROSE BEADS Cartridges TECHNICAL SPECIFICATIONS BEAD GEOMETRY

Spherical, Fine: ~ 20 - 50 µm

CROSSLINKED

Highly crosslinked 6 % agarose

MAX. BACK PRESSURE FLOW RATE CARTRIDGE PORTS

0.3 MPa, 3 bar Recommended: 1 ml / min. Maximum: 4 ml / min Inlet port & outlet port: 10 – 32 (1/16´´) female

PRODUCT NAME CAT. No. INFORMATION

Protein A Rapid RunTM Cartridges 6RRFPA-Ctg1-2 (two cartridges with 1 ml of resin each one) 6RRFPA-Ctg1-5 (five cartridges with 1 ml of resin each one) ~3 mg rProtein A / ml medium ~ 25 mg human IgG / ml medium

ANTIMICROBIAL AGENT STORAGE TEMPERATURE

ABT offers the option to prepack empty cartridges. See page 43.

Protein A Rapid RunTM Cartridges are used to purify classes, subclasses and fragments of immunoglobulins from cell culture media and biological fluids. • Easy changeover: no need for optimization or protocol change. • Great adaptability: Cartridges suitable for MPLC, FPLCTM, ÄKTA design TM, batch, gravity, peristaltic pump & syringe. • High purity achieved in one purification standards.

20% Ethanol 2 - 8º C

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AFFINITY CHROMATOGRAPHY GST PURIFICATION Low Pressure GLUTATHIONE AGAROSE BEADS Purification of proteins is a vital part of research. Impure extracts generally contain a wide range of proteins with diverse biological functions and different chemistry, which need to be separated. Fusion proteins expressed from pGEX vectors contain a Glutathione S-transferase (GST) moiety and can therefore be purified to near homogeneity by affinity chromatography on glutathione (γ-glutamylcysteinylglicine) as a substrate to inactivate toxic small molecules via formation of mercapturic acid. Because the affinity of GST for its substrate is in the submillimolar range, immobilization of glutathione on an agarose matrix makes a highly efficient affinity chromatography resin.

Glutathione has been covalently linked for use in affinity purification of glutathioneS-transferase (GST) and GST fusion proteins. This product provides a one step purification method and permits rapid, mild and highly selective purifications of proteins containing glutathione binding sequences. Bound GST –fusion proteins are easily displaced from the resin by elution with buffers containing reduced glutathione. ABT offers the Glutathione beads with an agarose percentage type (4% Agarose). The product range covers 2 different formats depending on the application type. FORMAT

PRODUCT

BINDING CAPACITY

APPLICATION

BULK RESINS (10, 100 ml)

GLUTATHIONE Agarose Resin

> 8 mg recombinant GST/ml gel

Batch Gravity MPLC FPLCTM

CARTRIDGES (5 X 1 ml)

GLUTATHIONE Agarose Cartridges

~ 10 mg recombinant GST/cartridge

Peristaltic pump Syringe MPLC FPLCTM ÄKTA designTM - 29 -


Low Pressure GLUTATHIONE AGAROSE BEADS

Resins & Cartridges

TECHNICAL SPECIFICATIONS

Glutathione Agarose resins for use in affinity purification of Glutathione-S-transferase (GST) and GST tagged fusion proteins.

BEAD (Geometry, size)

• Easy changeover: no need for optimization or protocol change. • Great adaptability: Bulk and Cartridges formats suitable for MPLC, FPLCTM, ÄKTA designTM, batch, gravity, peristaltic pump & syringe. • High purity achieved in one purification step, comparable to market standards.

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X: Product Quantity (10 or 100 ml).

No

AGAROSE % LIGAND PRODUCT NAME

• For all kind of proteins: small, large protein complex low expression rate proteins.

Spherical, Standard: ~50-150 µm

CROSSLINKED

CAT No. BINDING CAPACITY ANTIMICROBIAL AGENT STORAGE TEMPERATURE

4% Glutathione, linked via sulphur atom GLUTATHIONE Agarose Resin

GLUTATHIONE Agarose Cartridges (5 x1ml)

4B-GLU-X

4B-GLU-Ctg 1-5

> 8 mg recombinant GST /ml gel

~ 10 mg recombinant GST /Cartridge 20% Ethanol 4 - 8º C


GTS PURIFICATION AFFINITY CHROMATOGRAPHY

TECHNICAL PERFORMANCE DATA

Experiment Clarified E.coli lysate containing recombinant GlutathioneS-Transferase, MW 36.100 Da was offered (under the same conditions) to different Glutathione Agarose cartridges.

ABT CARTRIDGE

COMPETITOR CARTRIDGE

12.2 (mg purified protein /cartridge)

11.8 (mg purified protein /cartridge)

PURITY 94.5%

PURITY 93.6 %

ABT-CARTRIDGE

COMPETITOR -CARTRIDGE

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ENZYME & ANTIBODY IMMOBILIZATION Immobilization is a technique that binds a biomolecule (enzyme, antibody, affinity proteins like Protein A or G) to a support giving high stability and making easier re-using the immobilized molecule. The choice of Glyoxal or Aminoethyl will depend on the biomolecule to be immobilized, the accessibility of the reactive groups and the direction/ orientation required for the binding to the support. The easiest strategy is to screen with the correct Test Kit, and decide on options.

GLYOXAL/AMINOETHYL BEADS

CNBr ACTIVATED BEADS

• Very stable.

• Unstable.

• High reproducibility.

• Low reproducibility.

• Ready to use.

• Previous hydration step required.

• Irreversible binding.

• Reversible binding.

• High yield.

• Low protein yield.

• Long shelf-life.

• Short shelf-life.

This covalent binding also confers a qualitative advantage compared to resins activated with CNBr:

HIGH AND VERY HIGH ACTIVATED RESINS

LOW AND VERY LOW ACTIVATED RESINS

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ACIDIC GROUPS Low Pressure

AMINOETHYL AGAROSE BEADS Test Kit Screening AMINOETHYL LOW TEST KIT Includes: 2ml VERY LOW Density AMINOETHYL 2ml LOW Density AMINOETHYL

CAT. No. COMMENTS

4BCL 6BCL

AMINOL-2 Immobilization for acidic groups (Asp, Glu)

AMINOETHYL COMPLETE TEST KIT Includes: 2ml VERY LOW Density AMINOETHYL 2ml LOW DensityAMINOETHYL 2ml HIGH Density AMINOETHYL 2ml HIGH Density AMINOETHYL

CAT. No.

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AMINOC-2

4BCL 6BCL 4BCL 6BCL


ENZYME & ANTIBODY IMMOBILIZATION

Bulk Resins ABT Aminoethyl resins allow covalent binding of agarose to acidic group amino acids of the target biomolecule.

TECHNICAL SPECIFICATIONS BEAD (Geometry, size)

Spherical, Standard: ~50-150 µm

• Different quantities (25 and 100 ml).

CROSSLINKED AUTOCLAVABLE

121º C, 30’

• Larger amounts available on request.

MATRIX ACTIVE GROUPS

Amino Groups

PRODUCT NAME

Yes

LOW Density AMINOETHYL 6BCL

VERY LOW Density AMINOETHYL 4BCL

HIGH Density AMINOETHYL 6BCL

HIGH Density AMINOETHYL 4BCL

6%

4%

6%

4%

15-25

3-6

40-60

40-60

DESCRIPTION

CAT. No.

VERY LOW Density AMINOETHYL 4BCL

4BCL-AVL4-X

LOW Density AMINOETHYL 6BCL

6BCL-AL0-X

HIGH Density AMINOETHYL 4BCL

4BCL-AH1-X

ANTIMICROBIAL AGENT

0.5M NaCl containing 0.02% thimerosal or 20% Ethanol

HIGH Density AMINOETHYL 6BCL

6BCL-AM3-X

STORAGE TEMPERATURE

2 - 8º C

AGAROSE% ACTIVATION DEGREE (µmol diaminoethyl/ml gel)

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High Pressure AMINOETHYL RAPID RUNTM AGAROSE BEADS

Bulk Resins

high flow and stability characteristics of our crosslinked beads. Aminoethyl Rapid Run agarose beads is a pre-activated resin Proteins and other molecules containing aldehyde groups can that combines the advantages of Aminoethyl resins with the be coupled directly to the resin. TM

TECHNICAL SPECIFICATIONS CROSS-LINKED

Highly crosslinked agarose

MATRIX ACTIVE GROUPS

Amino Groups

AGAROSE% PRODUCT NAME

6% HIGH Density AMINOETHYL 6 RAPID RUNTM

VERY LOW Density AMINOETHYL 4 RAPID RUNTM

HIGH Density AMINOETHYL 4 RAPID RUNTM

VERY LOW Density AMINOETHYL 6 RAPID RUNTM

High Flow Minimum distortion of immobilized Biomolecule. Exclusion Limit ~4 x 106

High Flow Multiple Binding points. High immobilized Biomolecule stability. Exclusion Limit ~4 x 106

High Flow Minimum distortion of immobilized Biomolecule. Exclusion Limit ~3 x 107

High Flow Multiple Binding points. High immobilized Biomolecule stability.Exclusion Limit ~3 x 107

High Flow Minimum distortion of immobilized Biomolecule. Exclusion Limit ~4 x 106. Recommended for packing cartridges

Spherical, Standard: ~50-150 µm

ACTIVATION DEGREE (µmol diaminoethyl/ml gel)

Spherical, Fine: ~20-50 µm

15-25

40-60

3-6

40-60

3-6

6RR-ALO-X

6RR-AM3-X

4RR-AVL4-X

4RR-AH1-X

6RRF-AVL4-X

ANTIMICROBIAL AGENT STORAGE TEMPERATURE - 36 -

6%

LOW Density AMINOETHYL 6 RAPID RUNTM

BEAD (Geometry, size)

CAT. No.

4%

X: Product Quantity (25 or 100 ml)

0.5M NaCl containing 0.02% thimerosal or 20% Ethanol 2 - 8º C


ENZYME & ANTIBODY IMMOBILIZATION

AMINO GROUPS Low Pressure

GLYOXAL AGAROSE BEADS Test Kit Screening

GLYOXAL TEST KIT Includes: 2ml LOW Density GLYOXAL 4BCL 2ml HIGH Density GLYOXAL 6BCL 2ml VERY HIGH Density GLYOXAL 6BCL

CAT. No. COMMENTS

2ml HIGH Density GLYOXAL 4BCL 2ml LOW Density GLYOXAL 6BCL

GLYOXK-2 Immobilization for basic groups ( Lys)

Bulk Resins ABT Glyoxal resins allow a covalent binding of agarose to lysine amino groups of the chosen biomolecule. • Different quantities (25 and 100 ml). • Larger amounts available on request.

(25 or 100 ml)

- 37 -


TECHNICAL PERFORMANCE DATA

BINDING CAPACITY (mg BSA/ ml gel)

ABT BEADS: Higher binding capacity and much faster kinetic binding of biomolecule (bovine serum albumin). - 38 -


ENZYME & ANTIBODY IMMOBILIZATION

High Pressure GLYOXAL RAPID RUNTM AGAROSE BEADS Bulk Resins

Proteins and other molecules containing amino groups can be coupled directly to the resin. A typical application of Glyoxal Rapid RunTM is based on antigen-antibody interactions with monoclonal antibodies as ligands.

Glyoxal Rapid Run agarose beads is a pre-activated resin that combines the advantages of Glyoxal resins with the high flow and stability characteristics of our highly cross-linked beads. TM

TECHNICAL SPECIFICATIONS CROSS-LINKED

Highly crosslinked agarose Agarose with some diols oxidized to aldehydes

BEAD (Geometry, size)

Spherical, Standard: ~50-150 µm

AGAROSE% PRODUCT NAME

ACTIVATION DEGREE (µmol diaminoethyl/ml gel)

CAT. No. ANTIMICROBIAL AGENT STORAGE TEMPERATURE X: Product Quantity (25 or 100 ml)

6%

Spherical, Fine: ~20-50 µm 4%

6%

4%

LOW Density GLYOXAL 6 RAPID RUNTM

HIGH Density GLYOXAL 6 RAPID RUNTM

LOW Density GLYOXAL 4 RAPID RUNTM

HIGH Density GLYOXAL 4 RAPID RUNTM

LOW Density GLYOXAL 6 RAPID RUNTM FINE

LOW Density GLYOXAL 4 RAPID RUNTM FINE

High Flow Minimum distortion of immobilized Biomolecule. Exclusion Limit ~4 x 106

High Flow Multiple Binding points. High immobilized Biomolecule stability. Exclusion Limit ~4 x 106

High Flow Minimum distortion of immobilized Biomolecule. Exclusion Limit ~3 x 107

High Flow Multiple Binding points. High immobilized Biomolecule stability. Exclusion Limit ~3 x 107

High Flow Minimum distortion of immobilized Biomolecule. Exclusion Limit ~4 x 106. Recommended for packing cartridges

High Flow Minimum distortion of immobilized Biomolecule. Exclusion Limit ~3 x 107. Recommended for packing cartridges

15-25

40-60

15-25

40-60

15 - 25

15 - 25

~15

6RR-GLO-X

6RR-GM3-X

4RR-GL0-X

4RR-GH1-X

4RRF-GLO-X

4BRRF-5L-X

6RRF-GLO-X

4% LOW Density GLYOXAL RAPID RUNTM 5 atoms High Flow Minimum distortion of immobilized Antibodies Exclusion Limit ~3 x 107. Recommended for packing cartridges

20% Ethanol 2 - 8º C

- 39 -


ACCESSORIES ABT offers single-use chromatography tools, which simplifies the use of the resins making the purification process a simple and rapid one.

Empty Mini Columns

Empty Spin Columns

Empty Columns

Serves as a tool for purification using small quantities of resin (100 – 250 µl) It is a single-use format for centrifuge purifications.

Allows working with small quantities of resin, around 50 to 100 µl. Are adequate for purifications with syringe (luer lock system) or by centrifugation.

Are adequate for working with gel volumes of 0.5 – 2 ml (Plastic Columns) or 2 – 6 ml (Plastic Columns XL).

- 40 -


ACCESSORIES EMPTY COLUMNS

TECHNICAL SPECIFICATIONS PRODUCT CAT. No.

PLASTIC MINI COLUMNS(*)

PLASTIC SPIN COLUMNS (**)

PLASTIC COLUMNS(*)

PLASTIC COLUMNS XL(*)

MC-100

SP-25

C-50

CXL-50

COLUMN/SPIN MATERIAL

Polypropylene

FRIT MATERIAL

Polyethylene

FRIT PORE SIZE

20 µm(*) / 35 µm(**)

CHEMICAL STABILITY CAPS CAPACITY (ml)

Stable in all commonly used reagents _

Top caps: Luer lock & screw cap End cap included

Top & end caps included

Top & end caps included

1.5

0.8

12

35

For single-use applications. Larger amounts of columns available on request.

- 41 -


ACCESSORIES

Empty Cartridges ABT offers single-use Empty Cartridges (1 ml) compatible to the common chromatography instruments.

ACCESORIES CARTRIDGES PRODUCT

QUANTITY

CAT. No.

COMMENTS

STOP PLUG (2)

10 / pack

SEB-10

For storage of the packed cartridge, Stop Plugs need be ordered separately.

SYRINGE CONNECTOR

1

SCEB-1

10-32 male / female luer connector. This adapter is used to connect syringes.

• Easy to pack. • Reduced cost. • Reproducible packing. The customer can use any type of chromatography media (> 20 µm) including the ABT range of products for packing the cartridges. We recommend packing using the bulk resins available from ABT.

- 42 -

Larger amounts available on request. ABT also provides service to pack resins.


ACCESSORIES EMPTY CARTRIDGES

TECHNICAL SPECIFICATIONS PRODUCT CAT. No. CONTAINS

5 Empty Cartridges (5 Cartridge Bodies and 10 End Plugs)(1) Polypropylene

FRIT MATERIAL

Polyethylene

FRIT PORE SIZE AVERAGE

12 µm

INNER COLUMN DIAMETER

6.2 mm

CHEMICAL STABILITY CAPACITY (ml)

2

EB-Ctg1-5

COLUMN MATERIAL

CONNECTIONS

1

EMPTY CARTRIDGES (1 ml)

Standard connection compatible to the common chromatography instruments (such as ÄKTA) Stable in all commonly used reagents 1 ml packed resin(2)

For storage of the packed cartridge, Stop Plugs need be ordered separately (see accessories Cartridges). The packed volume depends on the type resin. As a guide, the packed volume should be 1.2 – 1.4 ml for agarose based spherical particles. The recommended packed volume is 1.3 ml for Rapid RunTM Fine Resins from ABT.

Note: After the chosen resin is packed in the cartridge, the cartridge can be readily stored and re-used many times. If longer bed height is needed, 3 cartridges can be inter-connected to give a final bed height of 10 cm. Larger amounts available on request.

- 43 -


EUROPE & WORLD

USA

C/ La Forja, 9, 28850 Torrej贸n de Ardoz, Madrid, SPAIN Phone (+34) 91 761 02 32/30 Fax (+34) 91 675 74 44 info@abtbeads.es

5008 West Linebaugh Ave. Suite 44 Tampa, FL 33624, USA Phone 813 - 908 - 2589 Fax 813 - 908 - 3190 techserv@abtbeads.com

www.abtbeads.es

www.abtbeads.com


Product Catalog