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Choose the direct route. Purify with a pro.

Amicon® Pro Purification System

EMD Millipore is a division of Merck KGaA, Darmstadt, Germany

Introducing the Amicon® Pro purification system
Traditional protein purification is a long process with many steps and multiple devices, often resulting in protein degradation and loss. Avoid the risks associated with sample transfer and reduce hands-on time when you bind, wash, elute and/or concentrate your protein in the all-in-one Amicon® Pro purification system. No matter what your workflow, the Amicon® Pro system delivers consistent, accurate sample preparation, resulting in more reliable recovery, uncompromised purity and easier data generation. Because of the extremely flexible, modular design of the Amicon® Pro system, you can configure the perfect device for your protein preparation. To guide you best, we’ve organized this brochure with respect to application, each of which has a corresponding Amicon® Pro device configuration.

Amicon® Pro Application

Components

Protocol Steps

Resin Guideline ≤200 μL packed resin1

Benefits3 • Speed • No sample transfer - No loss • Improved yield • Range of sample volumes can be processed • Single elution -no fractions • Speed • No sample transfer - No loss

PURIFICATION With buffer Exchange device + • Bind exchange Amicon® Ultra filter • Clear and Wash and/or • Elute/Concentrate concentration +/- Buffer Exchange Purification only Exchange device • Bind • Clear and Wash • Elute

≤1000 μL packed resin2

DEPLETION OR ENRICHMENT Exchange device + • Bind ≤200 μL packed Amicon® Ultra filter • Deplete/Concentrate resin1 +/- Wash/Concentrate +/- Buffer Exchange DIAFILTRATION Exchange device + • Buffer Exchange Amicon® Ultra filter +/-Concentrate not applicable

• Speed • Improved activity of purified protein • Speed • No sample transfer - No loss • Improved yield

Featured application: Antibody labeling

Exchange device + • Buffer Exchange not applicable Amicon® Ultra filter • Labeling Reaction • Wash and Concentrate +/- Buffer Exchange

1 The assay capacity is dictated by the processing limitations of the Amicon® Ultra 0.5 mL device. (approximately 1 mg pure protein) 2 The Bind-Wash-Elute protocol is linearly scalable (50-1000 µL). Higher resin volumes require greater centrifugation speeds and duration to ensure filtrate clearance. 3 Benefits are relative to other current methods.

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PURIFIC ATION
Why is traditional protein purification so complicated?
Traditional resin-based purification involves adding sample to the resin, washing the resin multiple times and collecting the eluate in multiple fractions. Sometimes, this eluate is still not ready for your next step, and subsequent concentration or buffer exchange is required. The Amicon® Pro purification system collapses the protein purification workflow into as few as three spins, in a single device. Let us show you how you can use the Amicon® Pro system to take the direct route to protein purification.

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Purification with buffer exchange and/or concentration
1400 1200 1000

Bind, wash, elute and concentrate all in one centrifugal device.
The Amicon® Pro device streamlines the affinity purification process by providing a single adaptable centrifugation unit capable of performing all steps in the affinity purification process. The device combines affinity-based spin column purification with downstream sample concentration and buffer exchange, eliminating the need for multiple sample transfers, thereby minimizing protein loss. The large reservoir reduces the need for multiple centrifugation steps. Attaching the Amicon® 0.5 mL filter during the elution step provides simultaneous concentration and the option of highly efficient diafiltration (>99%) in a single spin. Combining these steps in a single device reduces the need for multiple pipetting steps, consumable devices and sample transfers, potentially minimizing protein loss.

Protein Yield (µg)

800

600 Incubate 400 Spin

Spin

Wash
0

200

Elute sample into collection tube
E1 (0.5 mL) E2 (0.5 mL) E3 (0.5 mL) Total (1.5 mL)

Spin

Spin Add resin and wash buffer. Add sample and resuspend resin.

E1 (1 mL)

E2 (1 mL)

Competitor 0.5 mL affinity-based spin column

Amicon® Pro Device

Collect filtrate and wash fractions (optional).

Add elution buffer and resuspend resin.

Eluted sample

BIND

Elute, don’t dilute. While three elution steps were required WASH ELUTEa single for the smaller affinity-based spin columns (left), spin in the Amicon® Pro device was sufficient to recover > 90% of resin-bound protein in an average of 45 µL final volume*.
* For this sample, the included Amicon® Ultra 0.5 mL filter was attached, so that the sample underwent simultaneous concentration during the elution step. The fraction’s final concentrated volume was, on average, 45 µL following a 15 minute spin.

Incubate Spin Wash Spin Add resin and wash buffer. Add sample and resuspend resin.

00

Collect filtrate and wash fractions (optional).

Attach Amicon® Add elution buffer Ultra device. and resuspend resin.

Add exchange buffer and spin (optional).

Remove Amicon® Ultra device, place collection tube over top, and invert. Spin to recover sample.

BIND

WASH

ELUTE AND CONCENTRATE

EXCHANGE BUFFER

Jump from lysate to concentrated, pure protein in a single device. Direct, no-transfer protein workflow for affinity purification, concentration and buffer exchange using the Amicon® Pro purification system. If using ≤ 200 µL packed resin, you can attach the Amicon® Ultra 0.5 mL device for simultaneous concentration during the elution step and optional buffer exchange. Invert the Amicon® Ultra 0.5 mL device and spin to collect your final sample.

COMPARE THE AMICON® PRO WORKFLOW WITH TRADITIONAL METHODS:
Purification + Concentration + Buffer Exchange Process time* No. of spins required Sample transfers
*Process time excludes 60 min. incubation for both protocols.

0.5 mL spin column + concentration device 67 minutes 12 1

Amicon® Pro purification system 29 minutes 6 0

4

10K

4 00

1 00

3 00

2 00

2 00

3 00

1 00

4 00

00

Spin

Elute sample into Amicon® Ultra device

Spin

Spin

10K

Spin

COLLECT

Purification only
Bind, wash, and elute all in one centrifugal device.
If you need to purify protein on a larger scale (using up to 1 mL of settled resin), eliminate tedious gravity columns and take the Amicon® Pro device for a spin. You’ll love the increased recovery and easier washing. Traditional gravity-driven columns can mean monotonous chromatography, analysis of multiple fractions and multiple chances for protein loss. Don’t take those chances—switch to Amicon® Pro centrifugal purification using your resin of choice.

Excellent recovery, regardless of scale.
Whether you are using a few microliters or a milliliter of resin, Amicon® Pro purification yields consistent recovery.
2.5

2.0 1.5 1.0 0.5 0
R² = 0.9989

Protein recovered increases linearly with respect to packed resin volume, offering flexibility in experimental scale.
1.0

Protein Recovered (mg)

0

0.2

0.4

0.6

0.8

Packed Resin (mL)

Better washing and recovery. Less pipetting.
Give your pipetter and your centrifuge a break! Achieve superior purity and excellent protein yield with less effort. Because of the large volume of the exchange device, you can wash with as much as 9 mL per spin, and then elute all of your protein in a single spin fraction. Compared to standard affinity spin columns that require up to three spins at each step, the Amicon® Pro device offers comparable performance in a fraction of the time.

100 Flowthrough Wash 1 Wash 2 Wash 3

80

60

40

One-spin wash efficiently clears nonspecifically bound protein using the Amicon® Pro system. In contrast, gravity columns and competitor spin columns require multiple washes to clear out nonspecifically bound protein.

% Total Protein

20

0

Gravity Column

Amicon® Pro device

Competitor Spin

Incubate Spin Spin Wash Spin Add resin and wash buffer. Add sample and resuspend resin. Collect filtrate and wash fractions (optional). Add elution buffer and resuspend resin. Eluted sample

Elute sample into collection tube

Spin

Direct, no-transfer workflow for affinity purification. For larger scale protein purification (using > 200 µL packed resin, for example), you can take advantage of the Amicon® Pro system’s efficient bind-wash-elute workflow to minimize your hands-on time.

BIND

WASH

ELUTE

COMPARE THE AMICON® PRO WORKFLOW WITH TRADITIONAL METHODS:
Purification Only Process time Steps required Sample transfers
Spin
Incubate Spin

0.5-2 mL gravity column* 125 minutes 18 3-8 (# of fractions collected)
Elute sample Amicon® Ultra device

Amicon® Pro purification system 65 minutes 6 0
Spin

00

Spin Add resin and wash buffer. Add sample and resuspend resin.

Collect filtrate and wash fractions (optional).

Attach Amicon® Add elution buffer Ultra device. and resuspend resin.

Add exchange buffer and spin (optional).

Remove Amicon® Ultra device, place collection tube over top, and invert. Spin to recover sample.

BIND

WASH

ELUTE AND CONCENTRATE

EXCHANGE BUFFER

10K

4 00

1 00

3 00

*Process includes setup, purification and sample gravity column, time to assess collected fractions is also included. Wash collection. In the case of the into

2 00

2 00

3 00

1 00

4 00

00

Spin

10K

Spin

5

COLLECT

DEPLETION/ENRICHMENT
Depletion with concentration in one step.
Proteomic analysis requires depletion of overrepresented species to detect less abundant biomarkers. Using albumin-binding resin with the Amicon® Pro system provides the perfect tool for rapid sample depletion. In just one step, your sample is enriched, concentrated, and ready for 2D electrophoresis, LC/MS or other downstream analyses.

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One-step depletion: the direct route to informative proteomics data.
Removing albumin and immunoglobulins from biological samples can help reveal less abundant proteins of interest in proteomic analyses. However, traditional methods for depletion can be time-consuming and may risk sample loss. Using albumin-binding resin with the Amicon® Pro system enables one-step depletion with simultaneous concentration, minimizing sample transfer and maximizing recovery.

Spin Spin Wash Spin Add resin. Attach Amicon® Ultra device, add sample and resuspend resin.
4 00 3 00 2 00 1 00
00 00

Downstream applications: functional proteomics

LC/MS

MW St Se d rum De ple t Wa ed sh

Add wash buffer.

Remove Amicon® Ultra device, place collection tube over top, and invert. Spin to recover sample.

COMPARE THE AMICON® PRO WORKFLOW WITH TRADITIONAL METHODS:
Depletion Process time Steps required Sample transfers 0.5 mL spin column + concentration device 125 min 12 1

10K

2 00 3 00 4 00

Spin

1 00

Depletion of Albumin/IgG from human serum with concentration

10K

Spin

2D Gels

One-step depletion enriches your protein sample for more informative proteomics data. After binding your sample to a depletion resin (i.e., Anti-albumin), attach the Amicon® Ultra filter prior to centrifugal passage of the unbound fraction for simultaneous sample depletion with concentration in one step. Invert the Amicon® Ultra 0.5 mL device and spin to collect your final sample. Your sample is ready for proteomic analysis.

Amicon® Pro purification system 95 min 5 0

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DIAFILTRATION
Desalt in a single spin.
Buffer exchange using dialysis or diafiltration is often required to make a protein sample compatible with specific downstream analyses. But dialysis or multi-step diafiltration is timeconsuming, risks sample loss and can require subsequent concentration. The Amicon® Pro device offers ground-breaking, gentle, single-spin diafiltration for simultaneous buffer exchange with concentration.

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Diafiltration
Whether you’re performing this step after elution from resin (see Purification section) or whether you’re performing diafiltration only, you’ll experience the same high diafiltration efficiency designed into the device.

The uniquely designed interface between the exchange tube tip and the Amicon® Ultra device enables greater than 99% buffer exchange in a single spin. Buffer exchange, as shown in this
diagram, was measured by the replacement of a lowmolecular weight dye (yellow) with clear buffer (black arrows); while a high-molecular weight dye (bright blue) was retained inside the Amicon® Ultra device.

The gentleness of dialysis with the efficiency of diafiltration.
Dialysis cassette + concentrator Process time Recovery Specific activity (signal/µg GST-LLP) 16 hours 51% 0.195 0.5 mL diafiltration device (3 spin) 50 min. > 95% 0.17 Amicon® Pro purification system 20 min. > 95% 0.199

Gentler buffer exchange = greater activity. Eluted Samples of GST-lambda protein phosphatase (LPP) buffer exchanged and concentrated using Amicon® Pro device showed greater specific activity and percentage recovery than when prepared with a dialysis cassette (plus concentrator device) or 0.5 mL diafiltration spin column.

Faster, more efficient desalting.

% ProteinRetention (n=4)

Advantages of reducing number of spins for buffer exchange
• Sample is not repeatedly • Less chance of protein

120

100

80

60

diluted and re-concentrated aggregation

40

20

• Decreased chance of

Efficient desalting with higher recovery in a fraction of the time required for cassette-based dialysis. One-step diafiltration with the Amicon® Pro system combines the diafiltration speed of large-volume centrifugal devices with the excellent exchange efficiency of the Amicon® Ultra filter.
Cassette-based dialyzer
(Brand P)

0

protein precipitation
% Salt removal Process time

• Maintains high protein activity • Saves time

(Brand V, 20 mL)

Centrifugal diafiltration device

(Brand V, 0.5 mL)

Centrifugal diafiltration device

Amicon® Ultra-0.5
(3-spin)

Amicon® Pro
(1 spin)

99.9 16 hours

88.2 15 min

99.9 45 min

>99.9 45 min

>99.9 15 min

COMPARE THE AMICON® PRO WORKFLOW WITH TRADITIONAL METHODS:
Diafiltration Process time Steps required Sample transfers dialysis (cassettes, tubing) + concentration device 17 hours 6 2 0.5 mL centrifugal diafiltration devices 45 minutes 4 0

Amicon® Pro purification system 15 minutes 2 0

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Featured Application
Antibody labeling and high-yield purification in as little as one hour.
The Amicon® Pro purification system provides the ideal processing tool for smallbatch antibody labeling. Antibody cleanup routinely involves the removal of BSA/ carrier, labeling, removal of unbound label, dialysis, and final concentration. Using standard dialysis, the procedure can take up to 3 days to complete. In contrast, the Amicon® Pro method requires just one hour. Also, thanks to the all-in-one format of the Amicon® Pro device, it eliminates loss due to sample transfers and can significantly improve recovery. With the flexibility of the Amicon® Pro purification system, you can label small amounts of antibody quickly and easily with reliable performance and superior yield.
Generate FITC-labeled antibody in one hour. What’s faster than labeling antibodies using other purification methods, and more economical than purchasing prelabeled antibodies? Using Amicon® Pro purification systems for antibody labeling.

GAPDH

Control aGAPDH-FITC Ab

Dialysis Method Labeled Ab

Amicon® Pro Device Labeled Ab

Step Buffer exchange FITC labeling Free FITC removal and buffer exchange Total time Antibody recovery

Dialysis-based buffer exchange pre/post labeling Overnight 3h Overnight 3 days 39%

Amicon® Pro purification system 15 min 30 min 15 min 1h 72%

COMPARE THE AMICON® PRO WORKFLOW WITH TRADITIONAL METHODS:
Ab Labeling* Process time Steps required Sample transfers Amount Ab required Dialysis-based buffer exchange pre/post labeling 3 days 11 3 1 mg 0.5 mL centrifugal diafiltration devices 5 hours 8 0 20 µg

Amicon® Pro purification system 1 hour 5 0 20 µg

* The antibody labeling protocol consists of four basic steps: antibody cleanup, the labeling reaction, unbound dye removal/buffer exchange, and concentration. The duration of the labeling reaction can vary dependent on individual reagent parameters. The process time presented here is based on biotinylation using the Innolink™ Biotin 354S reagent (Cat. No. 203119, EMD Millipore)

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Ordering Information
To choose the appropriate Amicon® Pro device, determine the molecular weight cut-off (MWCO) of your protein of interest and your desired affinity purification scheme. For convenience and ease of use, the Amicon® Pro purification kits contain devices, reagents and buffers optimized for twelve reactions. These kits are ideal for affinity purification of tagged recombinant proteins, antibody purification and depletion.
Amicon® Pro Purification Kits 12/pk Includes reagent kit (resin & buffers) Amicon Pro Affinity Concentration Kit Ni-NTA
® ®

Reagent Kit Only ACR5000NT ACR5000PA ACR5000PG ACR5000GS

MWCO 3,000 ACK5003NT ACK5003PA ACK5003PG ACK5003GS 10,000 ACK5010NT ACK5010PA ACK5010PG ACK5010GS 30,000 ACK5030NT ACK5030PA ACK5030PG ACK5030GS MWCO 3,000 ACS500302 ACS500312 ACS500324 10,000 ACS501002 ACS501012 ACS501024 30,000 ACS503002 ACS503012 ACS503024 50,000 ACS505002 ACS505012 ACS505024 100,000 ACS510002 ACS510012 ACS510024 50,000 ACK5050NT ACK5050PA ACK5050PG ACK5050GS 100,000 ACK5100NT ACK5100PA ACK5100PG ACK5100GS

Amicon Pro Affinity Concentration Kit Protein A Amicon® Pro Affinity Concentration Kit Protein G Amicon® Pro Affinity Concentration Kit GST Amicon® Pro purification system – No Reagents Included  Amicon Pro Purification System Trial Pack 2/pk
®

Amicon® Pro Purification System 12/pk Amicon Pro Purification System 24/pk
®

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Components, features and benefits of the Amicon® Pro purification system
Only one device required for sample prep workflow, including: Gentle and efficient buffer exchange Broad reagent compatibility

• Purification • Concentration • Desalting / buffer exchange • No need to transfer • Better protein binding

• >99.99% (1000-fold)

• Agarose resin kits optimized
for the Amicon® Pro device >10 µm in diameter

desalting in a single spin • Maintains protein activity

• Compatible with other resins • Chemically compatible with
most common buffers

Superior sample recovery
with packed agarose resin compared to batch mixing

Collection Tube Cap Exchange Device Cap Exchange Device
Larger size holds larger wash volumes Unique tip fits into Amicon Ultra® 0.5 mL filter Enables single spin buffer exchange Easy-to-label cap can be used in place of collection tube cap in centrifuge

Ergonomic, less pipetting

• No need to transfer

Simultaneous concentration capability

• Elution without dilution • Consistent recovery • Saves time

Large reservoir saves time

Consistent concentration with the Amicon Ultra® 0.5 mL filter Provides elution without dilution Enables gentle diafiltration buffer exchange

Amicon® Ultra 0.5 mL Filter Amicon® Ultra 0.5 mL Filter Collection Tube
Reverse spin sample collection Easy, efficient, reproducible

• Holds wide range of volumes • Reduces spin steps • Reservoir cap can be labeled

Large volume 50 mL tube No need to empty between spins

Collection Tube

to reduce time and confusion

To Place an Order or Receive Technical Assistance
In the U.S. and Canada, call toll-free 1-800-645-5476 For other countries across Europe and the world, please visit: www.emdmillipore.com/offices For Technical Service, please visit: www.emdmillipore.com/techservice

www.emdmillipore.com/offices
EMD Millipore, the M mark, Direct Detect, PureProteome, YeastBuster, D-Tube, Overnight Express, CytoBuster and rLysozyme are trademarks and Amicon, BugBuster, Benzonase, ProteoExtract, PopCulture, SNAP i.d., ZipTip and Milli-Q are registered trademarks of Merck KGaA, Darmstadt, Germany. Trademarks belonging to third parties are the properties of their respective owners. Lit No. PB1502EN00 BS-GEN-13-08057 04/2013 Printed in the USA. ©2013 EMD Millipore Corporation, Billerica, MA, USA. All rights reserved.

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