Antibody Coupling Kit [Size: 50 Reactions]

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Application: Antibody Coupling

Particles: Not included in this kit. This kit is for magnetic, polystyrene and silica particles

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

  • The Activation Reagent in this kit can be used on any surface with any antibody and protein

  • Less hazardous chemicals to work with than NHS/EDC, the Activation Reagent in this kit is water based

  • Easy to use: You simply pipette the Activation Reagent on your surface to activate.

  • Promotes monolayer binding which increases antibody functionality

  • Reduces aggregation

  • Reduces interference

  • Increases uniformity between experiments

Product Description

Anteo’s ready-to-use Antibody Coupling Kit for magnetic, polystyrene and silica particles is a less hazardous alternative to covalent NHS/EDC chemistry. No messy pre-preparation of reagents required. Simply pipette Anteo's water-based Activation Reagent onto your chosen particles to activate. Hands-on time for surface activation and coupling is 60 minutes.

                        

With this kit, antibodies assemble in the correct orientation (monolayer binding) which 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.

 

Frequently Asked Questions – Antibody Coupling Kit (Ab Coupling Kit)

 

 

Q: How long does surface activation and coupling take with the Antibody Coupling Kit?

A: Hands on time for surface activation and coupling is 60 minutes.


 

Q: Is the Antibody Coupling 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: What type of linkage is formed?

A:  Our technology is built upon chelation and coordination chemistry with a binding strength equivalent to traditional covalent methods. The resulting activated surface functions as a double-sided velcro, whereby it strongly binds to your selected surface while allowing the gentle, yet strong binding of antibodies to the activated surface.


 

Q: What Antibodies are compatible with this kit?

The kit has been tested with the following antibodies: Mouse IgG, Mouse IgM, Rabbit IgG, Human IgG, Human IgA. The biggest advantage when using our kit is that you don't need to know anything about your antibody subclass to get accurate results and remove lengthy R&D steps before running your experiment. The flexibility to bind any antibody to your chosen bead without pre-treating your Antibody is finally here!

 

 

Q: What is different about Anteo’s Antibody Coupling Kit?

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

 

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

 

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: What particles are compatible with this kit?

A: Antibody Coupling Kit 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: Yes. Our Antibody Coupling Kit has been tested with antibodies from a variety of species including mouse, rabbit, goat and sheep

 

Q: What Antibodies are compatible with this kit?

The kit has been tested with the following antibodies: Mouse IgG, Mouse IgM, Rabbit IgG, Human IgG, Human IgA. The biggest advantage when using our kit is that you don't need to know anything about your antibody subclass to get accurate results and remove lengthy R&D steps before running your experiment.

The flexibility to bind any antibody to your chosen bead without pre-treating your Antibody is finally here!


 

Q: Can monoclonal and polyclonal antibodies be used?

A: Yes. Our Antibody Coupling 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 on 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.

 

Q: What Antibody concentration should be used?

The suggested coupling range for this kit is 25 to 100 µg/mg of particles. The particles are activated and coupled at 10 mg/mL so this would mean that the final antibody concentration range is 250 µg/mL to 1,000 µg/mL. It is suggested that the optimum concentration is determined for your antibody, as too much or too little antibody 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 antibody and if the antibody 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 antibody 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 will 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 antibody solution. Zeba™ Spin Desalting Columns (Thermo Fisher) work well without loss or dilution of the antibody. 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.

 

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 antibody- coupled particles?

Stability of the antibody-coupled particles will depend on the antibody 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.

 

Q: What is the procedure for co-coupling two or more antibodies to the particles?

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

 

[Antibody 1]

[Antibody 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

 

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 magnetic, polystyrene and silica 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 VMPABMP
Brand Anteo
Shipping Weight 0.2500kg
Shipping Width 0.175m
Shipping Height 0.080m
Shipping Length 0.160m
Shipping Cubic 0.002176m3

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