Magnetic Separation Coupling Kit [Size: 50 Reactions]

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Application: Use this kit to develop novel applications on magnetic particles - from antibody coupling, to antigens, peptides, carbohydrates, viruses, bacteria and cell surface markers.

Particles: Not included in this kit. This kit is for all magnetic particles containing including Carboxyl functionalisation

For Best Results: You will require a sonication bath to use this kit

Base: Water-based Activation Reagent is included in this Kit

Product Highlights:

  • Hand on time for surface activation and coupling is 60 minutes

  • Essential for exploratory work, achieve results when testing with unknown proteins

  • Couple any ligand to your particle of choice

  • Allows the development of multi-functional coupled particles with easy titration of ligand coupling concentration

  • No pre-treatment of proteins or antibody is required to achieve results

  • An alternative to covalent NHS/EDC and passive binding the Anteo Kit is easy to use, while reducing damage to antibodies

  • Less hazardous chemicals to work with than NHS/EDC, Anteo’s Activation Reagent is water based

  • Easy to use: Simply pipette the Activation Reagent on your surface to activate

  • Promotes monolayer binding which increases antibody functionality

  • Reduces aggregation and interference

  • Increases uniformity between experiments

  • Supports multi-functional protein separation

Product Description

Reduce labour costs by using our ready-to-use Magnetic Separation Coupling Kit. No messy pre-preparation of reagents required. Simply pipette Anteo's water-based Activation Reagent onto your chosen particle to activate. Hands-on time for surface activation and coupling is 60 minutes.

 

This kit greatly reduces the risk of antibody damage, leading to less antibodies required for the experiment, increased antibody functionality, reduced interference and increased uniformity between experiments.

 

This Anteo kit is also essential for exploratory work. Removing R&D steps required for this type of work, this Kit enables scientists to achieve results even when testing with unknown proteins.

 

The Anteo Magnetic Bead Separation Kit is compatible with Carboxyl beads to give scientists a cost effective option, while supporting their experiments with all the benefits that Anteo’s technology provides.

 

 

Frequently Asked Questions – Magnetic Separation Coupling Kit

 

 

Q: What is different about Anteo’s Magnetic Separation Kit?

A: The answer lies in Anteo’s Patented Technology:

 

1. Is very strong yet flexible, allowing the user to bind any ligand to their chosen particle without pre-treating the ligand.

 

2. Promotes monolayer binding: meaning antibodies assemble in the correct orientation reducing the amount of damage leading to increased functionality of antibodies. As a result, less antibody is required supporting a cost-efficient solution per sample.

 

3. Reduces aggregation: The activation reagent (included in the kit) contains a small amount of optimised surfactant, which reduces aggregation due to hydrophobic forces. It also creates a positively charged surface improving particle handling.

 

4. It is built upon chelation and coordination chemistry, which finally gives researchers the choice to use another method apart from passive and covalent bindings.

 

Q: Is the Magnetic Separation Kit easy to use?

A: Yes. Simply pipette Anteo’s Activation Reagent onto your chosen surface. The reagent is water based and all of the buffers you need from surface activation to coupling are provided.

 

Q: Which applications can be used with Magnetic Separation Kit?

A: Magnetic Separation techniques cover a broad range of applications, including:

  • Bioseparations (protein, cell, purification, binding partners)

  • Research, Development and discovery

  • The ease of use of magnetic coupling and handling allows quick development of novel techniques and discovery of new markers and target binding partners.

 

Q: How is your technology different to other competitives?

A:  Our technology is built upon chelation and coordination chemistry with a binding strength equivalent to traditional covalent methods. Our technology utilises metal polymer complexes. It allows multi-valent binding of the target antibody through chelation to the electron donating groups of the ligand to be coupled. This results in a gentle yet strong approach to coupling ligands.  

 

In contrast, covalent chemistries offer strong bonds that may damage the antibody structure.  Both passive adsorption and covalent chemistries often suffer from loss of activity due to the antibody structure being deformed or the active site of the antibody being shielded. The Anteo chelation coupling technology is also water based and stable so does not require fresh preparation, or the use of toxic chemicals used in some covalent methodologies.

 

Q: What particles are compatible with this kit?

A: Magnetic Separation Kit is compatible with carboxylated magnetic, polystyrene and silica particles ranging from 200 nm to 3 µm. The following table shows tested particles:

 

Supplier

Size

Material

Magnetic

 

Agilent LodeStars Carboxyl

2.7 µm

PS

Magnetic

Micro Particles

Bangs ProMag (PMC3N)

2.8 µm

PS

Magnetic

JSR Micro Magnosphere (MS300/Carboxyl)

3 µm

PS

Magnetic

Merck Millipore Estapor (M1-200/20)

2 µm

PS

Magnetic

ThermoFisher Dynabeads M-270 Carboxylic Acid

2.6 µm

PS

Magnetic

Allrun AllMag (PM3-050)

1 µm

PS

Magnetic

Sub-Micron Particles

Bangs ProMag (PMC1N)

1 µm

PS

Magnetic

GE Sera-Mag SpeedBeads

1 µm

PS

Magnetic

JSR Micro Magnosphere (MS160/Carboxyl)

1.5 µm

Hydrophilic polymer

Magnetic

JSR Micro Magnosphere (MX100/Carboxyl)

1.1 µm

Hydrophobic polymer

Magnetic

Merck Millipore Estapor (EM1-100/40)

1 µm

PS

Magnetic

PerkinElmer Chemagen M-PVA (C21)

1 µm

PVA

Magnetic

ThermoFisher Dynabeads MyOne Carboxylic Acid

1 µm

PS

Magnetic

ademtech Carboxyl-Adembeads 200nm

200nm

PS

Magnetic

Nano Particles

ademtech Carboxyl-Adembeads 500nm

500 nm

PS

Magnetic

Allrun AllMag (PM3-020)

200 nm

PS

Magnetic

Merck Millipore Estapor (M1-020/50)

200 nm

PS

Magnetic

Merck Millipore PureProteome Carboxy FlexiBind

300 nm

PS

Magnetic

Microparticles Polystyrene particles (PS-MAG-COOH-S2632)

350 nm

PS

Magnetic

ThermoFisher Blue Colored Carboxylate-modified (DB1040CA)

400 nm

PS

Non-Magnetic

 

Q Can antibodies from different species be used?

A:  Magnetic Separation Kit and following it’s basic protocol and conditions, a range of proteins covering several species and isotypes of antibodies, as well as bacterial recombinant proteins can be successfully coupled to Anteo activated particles with minimal optmisation. Also different proteins have different affinities for Anteo coupling, and in using this kit can be titrated to produce multifunctional particles.

 

 

Q: What proteins are compatible with this kit?

A panel of different proteins was tested to cover a wide range of immunoglobulin isotypes (IgA, IgG and IgM) and species sources (mouse, rabbit and human), as well as bacterial recombinant proteins (Protein
A, Protein G, Streptavidin) to highlight the flexibility of Anteo technology.  All of the tested proteins were coupled all at the same conditions without any consideration for protein size, sequence, tertiary structure, and subsequent affinity for the Anteo activated surface

 

The panel of coupled proteins suggests a range of proteins covering natural and recombinant proteins, sourced from mammals or bacteria; and cover single- and multi-domain proteins.  The fact that all these diverse proteins all coupled to some degree allows a starting point that the researcher can use knowing that it works, requiring minimal optmisation of the coupling, allowing more time spent on the actual application, making the Anteo Magnetic Separation Coupling Kit an excellent tool for research and development exploratory work.

 

Q: Can I produce multifunctional coupled magnetic particle using this kit?

Yes. By doing a simple titration of proteins to optimise the protein coupling onto the particles, it can be shown that this can also apply to multiple proteins.  Doing a simple titration of a mixture of 2 proteins (for example Streptavidin and antibody) using an approximate maximal protein concentration (which will depend on the binding capacity of the particles used) easily allows the generation of a multifunctional coupled magnetic particle that can have the ratios of each protein on the particle as the researcher desires.

 

Q: What is the procedure If I wan to co-couple two or more antibodies to the particles?

The mixture of your ligands for co-coupling can be added in the same way as for a single ligand, within the coupling concentrations recommended. As different types of ligand will bind at varying rates, it is recommended to titrate both, keeping the total concentration the same. See table below for example.

 

[Ligand 1]

[Ligand 2]

[Total]

0 µg/mL

200 µg/mL

200 µg/mL

25 µg/mL

175 µg/mL

200 µg/mL

50 µg/mL

150 µg/mL

200 µg/mL

75 µg/mL

125 µg/mL

200 µg/mL

100 µg/mL

100 µg/mL

200 µg/mL

125 µg/mL

75 µg/mL

200 µg/mL

150 µg/mL

50 µg/mL

200 µg/mL

175 µg/mL

25 µg/mL

200 µg/mL

200 µg/mL

0 µg/mL

200 µg/mL

 

Q: Can monoclonal and polyclonal antibodies be used?

A: Yes. Our Magnetic Separation Kit has been developed for use with both monoclonal and polyclonal antibodies.

 

 

Q: What should be done if aggregation is observed?

A: The activation reagent (included in the kit) contains a small amount of optimised surfactant which reduces aggregation due to hydrophobic forces. It also creates a positively charged surface improving particle handling.

In a case where some aggregation is observed, vortex to resuspend and sonicate for 5 min. If aggregation is still observed repeat the process.

 

 

Q: What sort of sonication can be used?

A: For the volumes in the IFU use a bath sonicator with fresh distilled and degassed water (minimum power 60W). For larger volumes a probe may be used. A probe sonicator would have to be optimised but general settings might be e.g. Sonics VC-750 Vibracell tip-sonicator 40% amplitude for 30 seconds.


 

Q: What if particles get stuck in the cap of the tube?

Spin the tube in a microcentrifuge for a short time before magnetic separation steps. This will ensure the particles are in the bottom of the tube. If the particles are not in solution, we also recommend a short sonication (1-5 minutes) in a bath sonicator.

 

 

Q: Can the protocol be scaled up?

Yes the protocol is directly scalable. Some of the incubation times may need to be optimised at larger scales. For example magnetic separation for a 5 mL reaction size would be different to a 1mL size. We suggest to leave the tube on the magnet until the supernatant clears, as time required would depend on type of beads and magnet used.  Other considerations may also require optmisation, such as the vessel type, separation and dispersion techniques.

 

Q: What Ligand concentration should be used?

The suggested coupling range for this kit is 25 to 100 µg/mg of particles. The particles are activated at 10 mg/mL and coupled at 5 mg/mL so this would mean that the final ligand concentration range is 250 µg/mL to 1,000 µg/mL. It is suggested that the optimum concentration is determined for your ligand, as too much or too little ligand may reduce the functionality of the coupled particles.

 

Q: What other chemicals can be used during coupling?

Refer to chemical compatibility table in the instructions for use for information on what can and cannot be used during coupling. It is suggested to use the included Coupling Buffer to dilute the ligand and if the ligand cannot be diluted more than 25% then buffer exchange may be required.

 

Q: What reagents need to be avoided during storage and coupling?

It is recommended to use the included Coupling and Storage Buffer. For more information refer to chemical compatibility table in the instructions for use.

 

Q: Do I have to desalt the antibodies if they come in PBS?

If the ligand cannot be diluted more than 25% (final phosphate concentration ≤ 2.5 mM) then buffer exchange or desalting will be required.  Phosphate Buffered Saline (PBS) is a metal chelator and can interfere with the coupling process.

 

Q: If so, what method do you suggest?

There are a number of options to buffer exchange, desalt, or dialyse the ligand solution. Zeba™ Spin Desalting Columns (Thermo Fisher) work well without loss or dilution of the ligand. Use the included instructions for buffer exchange.

 

Q: What magnetic separators do you use?

The magnetic separator used during the development of this kit is the DynaMag-2 available from Life Technologies (Cat No. 12321D). Other magnets may be suitable as long as the supernatant achieves complete functional clearing. Anteo may be selling our own magnets as well?

 

Q: Can the particles be stored frozen for later use?

It is not recommended to freeze the particles as this can cause irreversible clumping or clustering. Repeat freezing and thawing particles may create cracks on the surface of the beads that result in release of iron from the beads and contamination of the sample.

 

Q: Can the activated particles be stored lyophilized (freeze dried) for later use?

After using our technology your activated particles can be lyophilised. A small number of magnetic microparticles have been lyophilised and subsequently resuspended and coupled including particles validated with this kit.

 

Q: How can the drying of particles be prevented?

Drying can be prevented by storing the particles in the supplied buffer in an airtight container at 2-8ºC.

 

Q: What is the stability of the activated particles?

The activated particles are stable for up to one year, when stored at 2-8ºC.

 

Q: What is the stability of ligand- coupled particles?

Stability of the ligand-coupled particles will depend on the ligand itself. Generally coupled particles are stable for up to one year, when stored in Storage Buffer at 2-8ºC.

 

Q: Do the particles require blocking after coupling and what blockers do you recommend?

It is highly recommended to block these particles using the Blocking Buffer included with the kit.

 

Q: Do the particles require quenching after blocking/coupling?

As our technology uses metal chelation as opposed to active esters, like NHS/EDC, Tosyl or Epoxy, quenching is not required.

In This Kit:

  • Particle Pre-treatment Solution
  • Particle Activation Solution
  • Particle Intermediate Solution
  • Coupling Buffer
  • Blocking Buffer
  • Storage Buffer

Particles: Not included in this kit. This kit is for all magnetic particles including Carboxyl particles

For Best Results: You will require a sonication bath to use this kit

Shipping: Ambient

Storage Temp: 2 – 8°C

Shelf life: 12 months from date of manufacture

SKU VMPMSMP
Brand Anteo
Shipping Weight 0.2500kg
Shipping Width 0.175m
Shipping Height 0.080m
Shipping Length 0.160m
Shipping Cubic 0.002240m3

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