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Coupling Kit
200 nm Magnetic Particles
AI-LNPCKMP-01.00
INSTRUCTIONS FOR USE
Coupling Kit
200 nm Magnetic Particles
Antibody Concentration: The concentration range for coupling
antibody is 25 - 100 µg/mg particles, with a determined maximal
binding capacity around 100 µg/mg particles. Antibody coupling
concentration is best optimised as this can vary depending on the
protein used.
This product is for Laboratory Use Only
Provided Materials
AMG Coupling Kits utilise Mix&Go™ technology to stably bind
biomolecules of interest to functionalised polymer magnetic
particles. Mix&Go allows proteins to bind faster and with more
functionality. AMG Coupling Kits provide enhanced loading
capacity for the capture of the target molecules and downstream
applications use. The particles are provided fully activated with
Mix&Go and are ready to use, with general buffer solutions
known to work for a majority of applications.
AMG Coupling Kits are suitable for a range of magnetic,
fluorescent
and
chemiluminescent
immunoassays
and
purification applications using biomolecules including proteins,
peptides, antigens and antibodies.
Product Description
Catalogue #:
A-LNPCKMP-10 (10 Reactions)
A-LNPCKMP-30 (30 Reactions)
Particle:
Concentration:
200 nm, Superparamagnetic
10 mg/mL (1% w/v solids)
Kit Storage:
Shelf life at 2°C - 8°C is 1 year.
do not expose the AMG Coupling Kit to
temperatures exceeding 60°C or freezing.
Component
Activated 200 nm Magnetic Particles
Coupling Buffer - Formulation C
Blocking Buffer - Formulation B
Storage Buffer - Formulation A
Catalogue Number
A-CMPLNMP-X
A-CMPCBC1-X
A-CMPBBB1-X
A-CMPSBA1-X
1.7 mL low binding microcentrifuge tubes
Additional Materials Required
Antibody
Pipettes
Magnetic separator for tubes
Tube rotator
Vortex mixer
Sonication Bath with fresh water (Min Power 60W)
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Before Starting
Note: Product contains ProClin® 300 as preservative. Product
is not guaranteed DNase, RNase or endotoxin free. The user
must determine the suitability of the product for specific uses.
Allow all reagents to come to room temperature (20°C - 25°C).
Refer to the AMG Coupling Kit process flowchart on page 3.
Particle Preparation
1.
2.
3.
4.
Safety Precautions
Standard precautions exercised when handling laboratory
reagents should be adhered to. Refer to the Coupling Kit MSDS
for safety precautions.
Product Compatibility
Assay Compatibility: Particles are compatible with the majority
of existing uses and protocols. This allows for easy substitution of
the AMG Coupling Kit into your application of choice.
Kit Reagent Compatibility: Pre-activated particles are stable
under the provided conditions, however the presence of certain
materials, at the time of coupling, will interfere with and reduce
the coupling efficiency. It is recommended to use the provided
Coupling Buffer and reduce the presence of these reagents
during coupling as much as practically possible. For information
on interfering materials visit the Anteo Technologies website
(www.anteotech.com).
Always determine optimal conditions for your protein of interest.
Temperature: Particles can be incubated at 4ºC overnight, or at
20ºC – 37ºC for shorter periods of time (e.g. 1 hour).
Vortex the stock particles on high for 10 seconds.
Sonicate the stock particles for 5 minutes.
Aliquot 100 µL (1 mg) of the particle suspension to a
1.5 mL tube.
Place the tube on the magnetic separator for up to 5
minutes.
 Helpful Hint:
When separating magnetic particles from supernatant, it
is recommended to put the tube containing the particles
on the magnetic separator until the supernatant is clear
(up to 5 minutes), and carefully remove the supernatant
so as not to disturb the particle pellet. Low strength
magnets will take longer to form a pellet or may leave
residue on the tube.
5.
6.
7.
Remove and discard the supernatant from the tube.
Remove tube from magnet and add 100 µL of Coupling
Buffer to the tube.
Resuspend the particles by vortexing on high for 10
seconds.
 Helpful Hint:
Particles may partially aggregate and appear to stick on
the tube at this stage. This is the normal process
whereby the surface is equilibrating to the Coupling
Buffer conditions for subsequent coupling of your protein.
8.
9.
Antibody Coupling Procedure
The following procedure is an example of how to couple antibody
to the particles and states only general conditions. Protocols
should be optimised to meet individual requirements. Additional
information may be found on the Anteo Technologies website
(www.anteotech.com).
www.anteotech.com
Page 1 of 3
Repeat above wash steps 4 - 7 once (2 washes total).
Place the tube on the magnetic separator for up to 5
minutes.
10. Remove and discard the supernatant from the tube.
11. Resuspend the particles in 100 µL of Coupling Buffer.
By vortexing on high for 10 seconds.
Copyright
©2015 Anteo Diagnostics Ltd. All Rights Reserved
Effective Date: 26/03/2015
Review Date: 26/03/2018
Coupling Kit
200 nm Magnetic Particles
AI-LNPCKMP-01.00
Antibody Coupling
Release Information
 Helpful Hint:
The intra- and inter-assay protein loading, with mouse IgG,
CV is assessed for every batch of particles manufactured,
with a %CV of <15% achieved for all batches.
Particles may aggregate during coupling. This may be
part of the normal process whereby the surface during
coupling is interacting with your protein of interest. There
may be a need for optimisation of the coupling
conditions. Particles can be dispersed by 5 minute
incubations in a bath sonicator.
12. Prepare 100 µL of antibody at the required final
concentration in Coupling Buffer in a fresh 1.5 mL tube.
13. Aspirate the prepared particles.
14. While mixing the antibody solution gently on a vortex,
add the particles drop wise
15. Vortex on high for 10 seconds, and sonicate for 5
minutes. This results in 200 µL total volume, and
particles are at 5 mg/mL.
16. Incubate for 60 minutes at room temperature on a tube
rotator, keeping the particles in suspension.
Blocking
Blocking is recommended for all particles, particularly those that
are not fully coupled with protein; and/or for particles that are to
be used in downstream applications where complex mixtures are
used. Blocking should be assessed depending on the end use
and if non-specific binding is encountered.
17. Vortex particles for 30 seconds.
 Helpful Hint:
Sonicate the particles for 5 minutes if aggregation is
observed.
18. Add 20 µL of the Blocking Buffer to the tube.
19. Incubate for 60 minutes at room temperature on a tube
rotator.
D. Particle Storage
20. Vortex particles for 30 seconds.
21. Place the tube on the magnetic separator for up to 5
minutes.
22. Remove and discard the supernatant from the tube.
23. Remove tube from magnet and add 200 µL of Storage
Buffer to the tube.
24. Resuspend the particles by vortexing on high for 10
seconds.
25. Repeat above wash steps 20 - 24 once (2 washes
total).
26. Place the tube on the magnetic separator for up to 5
minutes.
27. Remove and discard the supernatant from the tube.
28. Remove tube from magnet and add 100 µL of Storage
Buffer to the tube.
29. Resuspend the particles by vortexing on high for 10
seconds.
30. Sonicate particles for 5 minutes.
31. Repeat above steps 28 - 29 once (2 washes total).
32. The particles are stably coupled with protein and ready
for use at a final concentration of 10 mg/mL. Particles
may be used immediately or stored at 4ºC.
Activated particles have been quantified using mouse IgG
binding capacity titration (refer to Table 1).
The binding capacity of functional reagent, in this case mouse
IgG, gives an indication of the amount of useable reagent that
can be captured by this product.
The monodispersity of particles refers to the level of
aggregation. As particles aggregate, the Polydispersity Index
(PdI) increases. Activated particles are assessed by Zetasizer
to determine the level of dispersion of the particles.
Table 1. Activated particles
Binding Capacity (mouse IgG, µg/mg particle)
Monodispersity (Polydispersity Index, PdI)
> 50
< 0.2
Technical Support
For questions regarding this product or for technical support
please refer to our website or contact us via one of the following
methods:
Anteo Technologies Pty Ltd
Unit 4, 26 Brandl Street
Eight Mile Plains
QLD 4113
Australia
Phone: +61 7 3219 0085
Fax: +61 7 3219 0553
Email: support@anteotech.com
www.anteotech.com
Other Mix&Go™ Scientific Products
For further information on other Mix&Go products please refer
to our website:
www.anteotech.com
All Anteo Products are sold as general purpose reagents for general laboratory and
research uses only. Anteo Products are not intended for diagnostic and/or therapeutic
purposes and no Anteo Product may be administered to humans. Anteo does not make
any representation or warranty that Anteo Products comply with all laws and
regulations that may be applicable to Customer’s use of any Anteo Product.
Anteo warrants that Anteo Products will conform to the specifications set forth on the
applicable Anteo Product Certificate of Analysis (“Anteo’s Limited Warranty”). The
Certificate of Analysis may be obtained by contacting Anteo. Anteo’s Limited Warranty
is wholly conditioned on the proper use of Anteo Products in the applications for which
they are intended, and Anteo makes no warranty (express, implied or statutory) for
Anteo Products that are modified; subjected to accident, misuse, neglect, unauthorized
repair or improper tampering, testing or storage; and/or used or handled contrary to
Anteo’s instructions as set forth on the Anteo Product label and/or in this insert.
Unless otherwise expressly provided in this document or in these Terms and
Conditions of Sale, Anteo disclaims all warranties, conditions and guarantees, whether
written, express, implied, statutory or otherwise, including but not limited to, the implied
warranties or guarantees of merchantability and fitness for particular purpose.
.
®
ProClin 300 is a registered trademark of Rohn and Haas Company.
 Helpful Hint:
If the particles have been stored, they should be
resuspended prior to use (step 29 - 30).
Storage and Stability
The unopened product should be stored at 2°C - 8°C. Remaining
materials should be retained in the supplied container and sealed
for future use. Product should not be exposed to temperatures
above 60°C.
www.anteotech.com
Page 2 of 3
Copyright
©2015 Anteo Diagnostics Ltd. All Rights Reserved
Effective Date: 26/03/2015
Review Date: 26/03/2018
Coupling Kit
200 nm Magnetic Particles
AI-LNPCKMP-01.00
AMG COUPLING KIT PROCESS FLOWCHART
Step 1-3:
Prepare and dispense activated particles
Step 4-11:
Wash particles into coupling buffer
Step 12:
Dilute antibody in coupling buffer
Step 13-14: Add particles to diluted antibody solution with
gently vortex mixing
Step 16:
Incubate 60 minutes
Step 17-18: Add blocking buffer to particle antibody solution
Step 19:
Incubate 60 minutes
Step 20-31: Wash particles into storage buffer
✓
www.anteotech.com
Page 3 of 3
Step 32:
Particles coupled and ready to use
Copyright
©2015 Anteo Diagnostics Ltd. All Rights Reserved
Effective Date: 26/03/2015
Review Date: 26/03/2018