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AMG Coupling Kit
1 µm Magnetic Particles
AI-SMPCKMP-03.00
INSTRUCTIONS FOR USE
Coupling Kit
1 μm Magnetic Particles
Protein Coupling Procedure
This product is for Laboratory Use Only
AMG Coupling Kits utilise Mix&Go technology to stably couple
proteins of interest to functionalised polymer activated 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
the majority of applications.
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).
Protein Concentration: Protein coupling concentration is best
optimised as this can vary depending on the protein used. A
recommended range for coupling is 25 - 100 µg protein/mg
particles.
Provided Materials
AMG Coupling Kits are suitable for a range of fluorescent and
chemiluminescent immunoassays and purification applications
using molecules including proteins, peptides, antigens, and
antibodies.
Component
Catalogue Number
Activated 1 µm Magnetic Particles
A-CMPAPMP-X
Coupling Buffer - Formulation A
A-CMPCBA1-X
Blocking Buffer - Formulation A
A-CMPBBA1-X
Storage Buffer - Formulation A
A-CMPSBA1-X
Product Description
Catalogue #:
A-SMPCKMP-10 (10 Reactions)
A-SMPCKMP-30 (30 Reactions)
Particle:
Concentration:
1 µm, Magnetic
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.
1.7 mL low binding microcentrifuge tubes
Additional Materials Required
Protein of interest
Pipettes
Magnetic separator for tubes
Tube rotator
Vortex mixer
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Note: This product contains ProClin 300 as a preservative. The
product is not guaranteed DNase, RNase or endotoxin free.
Purchaser must determine the suitability of the product for
specific uses.
Before Starting
Safety Precautions
 Helpful Hint:
Standard precautions exercised when handling laboratory
reagents should be adhered to. Refer to the AMG Coupling Kit
MSDS for safety precautions.
Allow all reagents to come to room temperature.
Particle Preparation
Refer to the process flow chart on page 3 for tips on
coupling.
When washing activated particles, it is recommended
to put the tube containing the particles on the magnet
until the supernatant is clear (at least 1 minute), and
carefully remove the supernatant so as not to disturb
the particle pellet.
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.
1.
2.
Kit Reagent Compatibility: Pre-activated particles are stable
under the provided conditions, however the presence of high
ionic strength (e.g. > 0.25M NaCl), PBS buffer and metal
chelators (e.g. < 0.05M EDTA) 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.
Coupled particles retain full functionality after treatment for 1 hour
with ≤5% Tween20, ≤0.05M EDTA, ≤1M NaCl, ≤8M Urea, ≤50%
DMSO, pH ranging from 2-11, temperatures up to 60°C and are
also stable for up to 10 minutes of continuous sonication.
Reagents that denature proteins, such as SDS-PAGE sample
buffer preparations with or without reducing agents such as DTT,
will disrupt the tertiary structure of the protein and reduce
functionality of the coupled particles.
3.
4.
5.
6.
 Helpful Hint:
Temperature: Particles can be incubated at 4ºC overnight, or at
20ºC - 37ºC for shorter periods of time (e.g. 1 hour).
Particles may partially aggregate at this stage. It
is normal for some particles to stick to the tube
while equilibrating to the Coupling Buffer
conditions. This will not interfere with subsequent
protein coupling steps.
7.
8.
9.
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©2015 Anteo Technologies Pty Ltd
All Rights Reserved
Resuspend the particles by vortexing for 20 seconds
on high speed.
Transfer 100 µL (1 mg) of the particle suspension to a
1.7 mL tube.
Place the tube on the magnetic separator for at least 1
minute, or until supernatant is clear of particles.
Remove and discard the supernatant from the tube.
Remove tube from the magnet and add 100 µL of
Coupling Buffer to the tube.
Resuspend the particles by vortexing for 20 seconds
on high speed.
Repeat the above wash steps, 3 – 6, once (i.e. two
washes of the particles).
Place the tube on the magnetic separator for at least 1
minute, or until supernatant is clear.
Remove and discard the supernatant from the tube.
Effective Date: 20/03/2015
Review Date: 20/03/2017
AMG Coupling Kit
1 µm Magnetic Particles
AI-SMPCKMP-03.00
10. Remove tube from the magnet and resuspend the
particles by vortexing for 20 seconds in 50 µL of
Coupling Buffer. Particles are now at 20 mg/mL (2x
concentration).
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 percent monodispersity decreases.
Activated particles are assessed by microscopy to determine the
level of monodispersed particles.
Protein Coupling
 Helpful Hint:
Refer to the Kit Reagent Compatibility notes above for
consideration of reagents that may affect coupling
efficiency.
11. Prepare 50 µL of protein at 2x the required final
concentration in Coupling Buffer in a fresh 1.7 mL tube.
12. Transfer 50 µL of the protein solution to the particle
solution tube from step 10, and vortex on high speed
for 10 seconds.
13. Incubate for 60 minutes at room temperature on a tube
rotator at ≥50 rpm, keeping the particles in suspension.
Blocking (OPTIONAL)
Blocking is suggested for particles 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.
If blocking is not required, proceed to “Particle Storage”
below.
14. Add 10 µL of the Blocking Buffer to the tube and vortex
on high speed for 10 seconds.
15. Incubate for 60 minutes at room temperature on a tube
rotator at ≥50 rpm, keeping the particles in suspension.
Particle Storage
16. Place the tube on the magnetic separator for at least 1
minute, or until supernatant is clear.
17. Remove and discard the supernatant from the tube.
18. Remove tube from the magnet and add 100 µL of the
Storage Buffer to the tube.
19. Resuspend the particles by vortexing on high speed for
20 seconds.
20. Repeat above wash steps 16 – 19 once (i.e. two
washes of the particles).
21. Place the tube on the magnetic separator for at least 1
minute, or until supernatant is clear.
22. Remove and discard the supernatant from the tube.
23. Remove tube from the magnet and resuspend particles
in 100 µL of Storage Buffer.
24. The particles are stably coupled with protein and ready
for use. The particle concentration is 10 mg/mL.
 Helpful Hint:
Depending on the protein of interest, particles may appear
aggregated when viewed under magnification. Aggregation
at this step can be reduced by bath sonication of the
particles or optimisation of protein concentration. Once
dispersed, the Storage Buffer should maintain dispersity.
Release Information
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.
Table 1. Activated particles
Binding Capacity (mouse IgG, µg/mg particle)
Monodispersity (%)
> 20
> 90%
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.
Particles coupled by the user should be assessed for individual
use and storage stability conditions, as this can vary depending
on the protein and conditions used.
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: [email protected]
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.
Activated particles have been quantified using mouse IgG binding
capacity titration (refer to Table 1).
www.anteotech.com
Page 2 of 3
Copyright
©2015 Anteo Technologies Pty Ltd
All Rights Reserved
Effective Date: 20/03/2015
Review Date: 20/03/2017
AMG Coupling Kit
1 µm Magnetic Particles
AI-SMPCKMP-03.00
AMG COUPLING KIT PROCESS FLOWCHART
Activated 1 µm
Magnetic Particles
Dilute protein (2x conc.) in
50 µL Coupling Buffer
(e.g. 2 mg/mL for 100 µg
protein/mg particles) to
fresh tube
Aliquot 100 µL
particles (1 mg) to
fresh tube
Equilibrate particles
into Coupling Buffer
with 2 washes and
resuspend at 2x
concentration (50 µL)
Add 50 µL protein
to 50 µL particles.
Vortex 20 seconds
Incubate 1 hour at
room temperature
with rotation
Place tube on
magnet for 1
minute and
remove
supernatant
Resuspend particle
pellet in 100 µL
Storage Buffer.
Vortex for 20
seconds
Repeat wash into
Storage Buffer again
and resuspend in
100 µL Storage
Buffer.
Particles ready to
use!
www.anteotech.com
Page 3 of 3
Copyright
©2015 Anteo Technologies Pty Ltd
All Rights Reserved
Effective Date: 20/03/2015
Review Date: 20/03/2017