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Purification Products


Info

Purification media: beads for column purification of your protein of interest.

These beads feature proprietary TargetLock chemistry, improving the chemical stability and durability of agarose beads upon repeated uses.

Products for Purification


Affinity Media


Blue Beads
Gelatin Beads
Heparin Beads

Tag-Binding Beads


Nickel Beads
Glutathione Beads

Activated Beads


Epoxy-Activated Beads
DVS-Activated Beads

Activated Carriers


Maleimide-Activated BSA
Maleimide-Activated OvA
Maleimide-Activated KLH

Antibody Purification


Ig-Thio-Capture Beads
Protein G Beads are produced using genetically engineered Protein G. Non-essential regions have been removed while leaving the IgG binding sites.
Protein A Beads are produced using genetically engineered Protein A. Nonessential regions have been removed while leaving the IgG binding sites.
Protein G
Protein A

Avidin Beads


Biotin-Capture Beads USBios modified Avidin provides a substantial improvement over native Avidin. High affinity to biotin is maintained, while background problems are minimized due to chemical modification on the protein.

Immuno Precipitation


Anti-Mouse IgG Beads Goat Anti-Mouse IgG (whole molecule) is affinity purified on Mouse IgG affinity column using IgG isolated from pooled normal Mouse sera. Eluted antibodies are further purified by Protein G column in order to separate IgGs from other classes of antibodies. Purified Antibodies are coupled to Sepharose Beads.

Protein Size Makers


Biotinylated Protein MW Markers

Affinity Chromatography


Different approaches for different situations
Affinity chromatography makes use of specific binding interactions between molecules. A particular ligand is chemically immobilized or “coupled” to a solid support so that when a complex mixture is passed over the column, only those molecules having specific binding affinity to the ligand are purified. Affinity purification generally involves the following steps: 1) Incubate crude sample with the immobilized ligand support material to allow the target molecule in the sample to bind to the immobilized ligand. 2) Wash away nonbound sample components from solid support, and 3) Elute and recover the target molecule from the immobilized ligand by altering the buffer conditions so that the binding interaction no longer occurs.

Depending on the type of molecule to be purified, its specificity, conditions under which it is stable, different types of ligands coupled to different types of support may be employed. As examples, three popular chromatographic media are: anti-mouse IgG coupled to sepharose beads for immune separation; Epoxy-Activated Beads, a preactivated support that contains high density of epoxy groups used for immobilization of various ligands; and heparin combined with sepharose beads, used for isolating heparin binding protein such as Antithrombin III, lipoproteins, various enzymes and DNA binding proteins. All three of these are based on the same support matrix, Sepharose CL-4B. But because of their different ligands, they are used for very different purposes.

The mouse beads have an anti-mouse IgG ligand bound to them, making them suitable for immunoprecipitation of mouse antibodies. [see protocol footnote 1] This can be done over a wide range of conditions, depending on your individual needs.

Table 1. Mouse Beads Specifications:

Matrix: Sepharose™ CL-4B
Coupling method: TargetLock
Coupling density: 0.7-1.2 mg/ml of Goat anti Mouse IgG
Mouse IgG binding capacity: > 0.6 mg/ml of beads
Mean bead size: 40 -165 um
pH Range: 3.0-11.0
Bead structure: Highly cross-linked spherical agarose, 4%
Max back pressure: 0.3 MPa, 3 bar
Max. flow rates: 4 ml/min/cm2
Recommended flow rate: 1-3 ml/min/cm2
Storage: 4°C in PBS pH 7.4, 0.3N NaCl added with 01% (w/v) Azide as a preservative.

Epoxy-activated beads have Oxiran as an active group. This makes them useful in immobilizing proteins, carbohydrates and various ligands via stable linkage to amino, thiol and hydroxyl groups. Coupling to hydroxyls is favored at a higher pH of 11-13, while Thiol-containing ligands are best coupled at pH 7.5-9.0.

Table 2. Epoxy-Activated Beads Specifications:

Matrix: Sepharose CL-4B
Active group: Oxiran
Active group density: 10–25 umole/ml
Bead size: 45-165 um
Bead structure: Highly cross-linked spherical agarose, 6%
Max back pressure: 0.3 MPa, 3 bar
Max. flow rates: 4 ml/min/cm2
Recommended flow rate: 1-2 ml/min/cm2
Stability of the matrix: pH 2-11

Heparin Beads use 20-35 Kd Heparin extracted from Porcine intestinal mucosa (Type I) as their binding ligand. They are used to isolate various heparin-binding proteins such as AntithrombinIII, enzymes, DNA binding proteins and lipoproteins. [see protocol footnote 2]

Table 3. Heparin Beads Specifications:

Matrix: SepharoseTM CL-4B
Coupling method: TargetLock
Heparin density: 0.7-1.2 mg/ml of 20-35 KD Heparin
Thrombin binding capacity: > 0.7 mg/ml of beads
Mean bead size: 40 -165 um
pH Range 3.0-11.0
Bead structure: Highly cross-linked spherical agarose, 4%
Max back pressure: 0.3 MPa, 3 bar
Max. flow rates: 4 ml/min/cm2
Recommended flow rate: 1-3 ml/min/cm2
Storage: 4°C in PBS pH 7.4, 0.3N NaCl added with 0.01% (w/v) Thimerosal as a preservative.

protocol footnote 1:

Immunoglobulin affinity-purification using Anti-Mouse Beads

A. Buffers required
Binding buffer (1L): Phosphate buffered saline (PBS) pH 7.4
Neutralization buffer (50ml): Tris 1M pH 8.0 Adjust pH to 8.0 with 1M HCl.
Wash buffer (200 ml): PBS pH 7.4 optional PBS plus 1% Triton X-100.
Elution buffer (100ml): 0.1M glycine buffer Adjust pH to 2.8 with 1N HCl.
Regeneration buffer (100ml): 0.1M glycine buffer Adjust pH to 2.4 with 1N HCl.
Storage buffer: PBS with 0.1% sodium azide as a preservative.

B. Sample preparation
Note: work at 0 °C to 4°C in order to minimize antibodies degradation whenever possible.
1. If serum is the source, dilute 5ml to 15ml final volume with Binding buffer at 4°C .
2. Centrifuge diluted serum supernatants for 15 minutes at 10,000xg at 4°C to sediment debris.
3. Filter supernatants through 0.45μm filter.

C. Column affinity-purification
Anti-Mouse Beads P9100-45
Note: Typically 5 ml of bed volume are used to purify antibodies from 0.5L of monoclonal antibody-containing culture, or 50 ml of x10 diluted serum.
1. Wash protein Anti-Mouse beads with 30 ml Binding buffer three times in 50 ml conical tube.
Allow the beads to sediment naturally for 5 minutes each time, and remove upper liquid phase with a pipette.
2. Use the washed beads to pack a 1-1.5 cm diameter column by pouring the beads into an empty column. After column preparation equilibrate the column with Binding buffer by washing with 5-10 column volumes. Recommended flow rates are 1-2 ml/min/cm2.
3. Bring the sample to room temperature. Apply the sample to column at a rate between 0.2ml/min to 0.5 ml/min, using a syringe or a pump. The total volume of the sample applied is not critical in most cases. Save the flow through fraction for SDS-PAGE analysis.
4. Wash with 20 column volumes of Wash buffer.
5. Prepare labeled microfuge tubes with 5% V/V of neutralization buffer in each tube. Place tubes on an ice bucket
6. Elute with Elution buffer into the labeled microfuge tubes, at flow rates of 1-2 ml/min/cm2.Two to four column volumes are usually needed for elution of the immunoglobulins. Use the elution buffer as blank when doing the quantitation of the target protein in eluted fractions.

D. Re-equilibration, Regeneration and Storage
1. Regenerate column by 10 column volumes of the regeneration buffer. Work quickly as exposure of column to pH 2.4 should be kept to a minimum.
2. Wash column with 10 bed volumes of Binding buffer, immediately at the end of elution step.
3. Storage conditions: Store column in a refrigerator with Storage buffer.

protocol footnote 2:

Salt purification of Heparin-binding proteins

1. Invert bottle several times to mix beads in storage buffer.
2. Pack column with 2-4ml beads
3. Equilibrate column with 10mM Tris-HCl pH 7.5. Matrix is robust -You may modify buffer to include reducing agents, urea, guanidine, sodium thiocyanate and detergents as needed.
4. Load your protein sample on column at 0.5-1.0 ml/min.
5. Wash column with approximately 30 volumes of the equilibration buffer.
6. Elute bound proteins by applying high salt buffer (10mM Tris-HCl pH 7.5 added with NaCl to 1.5N or use other common salts such as KCl and Ammonium sulfate).
7. Wash column with 20 volumes of ddH2O to regenerate it.
8. Equilibrate column with 10mM Tris-HCl pH 7.5.
9. Store column at 4°C in PBS pH 7.4, 0.3N NaCl added with 0.01% (w/v) Thimerosal as a preservative.
Epitope Tag Antibodies for Use in Protein Purification ...

 



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