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

Choose the direct route.Purify with a pro.Amicon® Pro Purification System

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Amicon® Pro Application Components Protocol Steps Resin Guideline Benefits3

PURIFICATION With buffer exchange and/or concentration

Exchange device + Amicon® Ultra filter

•Bind •Clear and Wash •Elute/Concentrate

+/- Buffer Exchange

≤200 μL packed resin1

•Speed•No sample transfer - No loss •Improved yield

Purification only

Exchange device •Bind •Clear and Wash •Elute

≤1000 μL packed resin2

•Range of sample volumes can be processed

•Single elution -no fractions

DEPLETION OR ENRICHMENT Exchange device + Amicon® Ultra filter

•Bind •Deplete/Concentrate

+/- Wash/Concentrate +/- Buffer Exchange

≤200 μL packed resin1

•Speed•No sample transfer - No loss

DIAFILTRATION Exchange device + Amicon® Ultra filter

•Buffer Exchange +/-Concentrate

not applicable •Speed•Improved activity of purified

protein

Featured application: Antibody labeling

Exchange device + Amicon® Ultra filter

•Buffer Exchange •Labeling Reaction •Wash and Concentrate

+/- Buffer Exchange

not applicable •Speed•No sample transfer - No loss •Improved yield

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.

Introducing the Amicon® Pro purification systemTraditional 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.

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PURIF IC 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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COMPARE THE AMICON® PRO WORKFLOW WITH TRADITIONAL METHODS:Purification + Concentration + Buffer Exchange

0.5 mL spin column + concentration device

Amicon® Pro purification system

Process time* 67 minutes 29 minutesNo. of spins required 12 6Sample transfers 1 0

Purification with buffer exchange and/or concentration

Elute, don’t dilute. While three elution steps were required for the smaller affinity-based spin columns (left), a single spin in the Amicon® Pro device was sufficient to recover > 90% of resin-bound protein in an average of 45 µL final volume*.

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.

* 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.

*Process time excludes 60 min. incubation for both protocols.

0

200

400

600

800

1000

1200

1400Pr

otei

n Yi

eld

(µg)

Competitor 0.5 mL affinity-based spin column Amicon® Pro Device

E1 (0.5 mL) E2 (0.5 mL) E3 (0.5 mL) E1 (1 mL) E2 (1 mL)Total (1.5 mL)

ELUTE AND CONCENTRATE

ELUTE

SpinSpinSpin

10K4

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10K4

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Spin

BIND WASH

Add resin andwash buffer.

Attach Amicon®Ultra device.

Remove Amicon® Ultra device, place collection tube over top, and

invert. Spin to recover sample.

Incubate

Wash

Spin

Spin

Spin

Add sample and resuspend resin.

Collect filtrate and wash fractions (optional).

BIND WASH

Add resin and wash buffer.

Incubate

Wash

Spin

SpinSpin

Add sample and resuspend resin.

Collect filtrate and wash fractions (optional).

Add elution buffer and resuspend resin.

Eluted sample

Add elution buffer and resuspend resin.

Add exchange bufferand spin (optional).

COLLECT EXCHANGE BUFFER

Elute sample into

collection tube

Elute sample into

Amicon® Ultra device

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.

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ELUTE AND CONCENTRATE

ELUTE

SpinSpinSpin

10K4

3

2

1

00

00

00

00

00

10K4

3

2

1

00

00

00

00

00

Spin

BIND WASH

Add resin andwash buffer.

Attach Amicon®Ultra device.

Remove Amicon® Ultra device, place collection tube over top, and

invert. Spin to recover sample.

Incubate

Wash

Spin

Spin

Spin

Add sample and resuspend resin.

Collect filtrate and wash fractions (optional).

BIND WASH

Add resin and wash buffer.

Incubate

Wash

Spin

SpinSpin

Add sample and resuspend resin.

Collect filtrate and wash fractions (optional).

Add elution buffer and resuspend resin.

Eluted sample

Add elution buffer and resuspend resin.

Add exchange bufferand spin (optional).

COLLECT EXCHANGE BUFFER

Elute sample into

collection tube

Elute sample into

Amicon® Ultra device

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.

COMPARE THE AMICON® PRO WORKFLOW WITH TRADITIONAL METHODS:Purification Only 0.5-2 mL gravity column* Amicon® Pro

purification systemProcess time 125 minutes 65 minutesSteps required 18 6Sample transfers 3-8 (# of fractions collected) 0

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.

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.

Purification only

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

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

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.

0

0.5

1.0

1.5

2.0

2.5

0 0.2 0.4 0.6 0.8 1.0

R² = 0.9989

Prot

ein

Reco

vere

d (m

g)Packed Resin (mL)

Gravity Column Amicon® Pro device Competitor Spin

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% T

otal

Pro

tein

FlowthroughWash 1Wash 2Wash 3

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

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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 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.

One-step depletion: the direct route to informative proteomics data.

COMPARE THE AMICON® PRO WORKFLOW WITH TRADITIONAL METHODS:Depletion 0.5 mL spin column

+ concentration deviceAmicon® Pro purification system

Process time 125 min 95 minSteps required 12 5Sample transfers 1 0

SpinSpin

10K4

3

2

1

00

00

00

00

00

10K4

3

2

1

00

00

00

00

00

Add wash buffer. Remove Amicon® Ultra device,

place collection tube over top, and invert. Spin to recover sample.

Add resin.

Spin Wash

Spin

Spin

Attach Amicon® Ultra device, add sample

and resuspend resin.

Depletion of Albumin/IgG from human serum with concentration

Downstream applications:functional proteomics

LC/MS

2D Gels

MW

Std

Seru

mDe

plete

dW

ash

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.

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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 time-consuming, 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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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.

% Salt removal 99.9 88.2 99.9 >99.9 >99.9

Process time 16 hours 15 min 45 min 45 min 15 min

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.

COMPARE THE AMICON® PRO WORKFLOW WITH TRADITIONAL METHODS:Diafiltration dialysis (cassettes, tubing)

+ concentration device0.5 mL centrifugal diafiltration devices

Amicon® Pro purification system

Process time 17 hours 45 minutes 15 minutesSteps required 6 4 2Sample transfers 2 0 0

Diafiltration

Advantages of reducing number of spins for buffer exchange

• Sample is not repeatedly diluted and re-concentrated

• Less chance of protein aggregation

• Decreased chance of protein precipitation

• Maintains high protein activity

• Saves time

The gentleness of dialysis with the efficiency of diafiltration.

Faster, more efficient desalting.

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 low-molecular weight dye (yellow) with clear buffer (black arrows); while a high-molecular weight dye (bright blue) was retained inside the Amicon® Ultra device.

0

20

40

60

80

100

120

Amicon® Pro(1 spin)

Amicon®Ultra-0.5(3-spin)

Centrifugaldiafiltration

device(Brand V, 0.5 mL)

Centrifugaldiafiltration

device(Brand V, 20 mL)

Cassette-baseddialyzer(Brand P)

% P

rote

inRe

tent

ion

(n=4

)

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.

Dialysis cassette + concentrator

0.5 mL diafiltration device (3 spin)

Amicon® Pro purification system

Process time 16 hours 50 min. 20 min.

Recovery 51% > 95% > 95%

Specific activity (signal/μg GST-LLP)

0.195 0.17 0.199

* 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)

Antibody labeling and high-yield purification in as little as one hour.The Amicon® Pro purification system provides the ideal processing tool for small-batch 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.

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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.

Featured Application

COMPARE THE AMICON® PRO WORKFLOW WITH TRADITIONAL METHODS:Ab Labeling* Dialysis-based buffer

exchange pre/post labeling0.5 mL centrifugal diafiltration devices

Amicon® Pro purification system

Process time 3 days 5 hours 1 hourSteps required 11 8 5Sample transfers 3 0 0Amount Ab required 1 mg 20 μg 20 µg

GAPDH

ControlaGAPDH-FITC Ab

Dialysis Method Labeled Ab

Amicon® ProDevice Labeled Ab

Step Dialysis-based buffer exchange pre/post labeling

Amicon® Pro purification system

Buffer exchange Overnight 15 min

FITC labeling 3 h 30 min

Free FITC removal and buffer exchange

Overnight 15 min

Total time 3 days 1 h

Antibody recovery 39% 72%

Protein ExpressionEMD Millipore’s portfolio of products for protein expression includes the best all-purpose bacterial strains and several specialty strains for difficult-to-express proteins, complemented by the powerful pET vector system and award-winning Overnight Express™ autoinduction systems. For mammalian expression, trust our transfection reagents and antibiotics, including the most potent G418 selection agent on the market.

Browse all our protein expression tools at www.emdmillipore.com/mobio and www.emdmillipore.com/biochemicals

Protein ExtractionEMD Millipore’s complete range of reagents and enzymes provide you with countless options so that you can put together the perfect extraction protocol for your particular cells and protein.

• Bugbuster® Master Mix• PopCulture® Extraction Reagent• YeastBuster™ Protein Extraction Reagent• CytoBuster™ Protein Extraction Reagent• Insect PopCulture® Extraction Reagent• ProteoExtract® Kits for Fractionation,

Peptide Enrichment, and Depletion• rLysozyme™ Enzyme• Benzonase® Nuclease• Protease and Phosphatase Inhibitors

and Inhibitor Cocktails

Browse all protein extraction products at www.emdmillipore.com/psp

Affinity PurificationUse our broad portfolio of tools for affinity purifying tagged recombinant proteins, antibody purification, nucleic acid purification, protein-protein interaction studies, and albumin/IgG depletion from serum or plasma.

Browse all affinity purification products at www.emdmillipore.com/psp

Protein DetectionExplore our novel protein detection platforms, including optimized reagents for chemiluminescent and fluorescent Western blots, as well as the SNAP i.d.® 2.0 system, which reduces blocking, washing and antibody incubation time from hours to minutes. And say goodbye to Bradford assays with the IR-based Direct Detect™ spectrometer!

Visit www.emdmillipore.com/westernblotting for details.

Protein Preparation for Mass SpectrometrySuccessful mass spectrometry requires extremely careful sample preparation, in order to avoid contamination and decrease the sample complexity. The ZipTip® is a 10 μL pipette tip with a bed of chromatography media fixed at its end with no dead volume. It is ideal for concentrating and purifying samples for sensitive analyses such as MALDI-ToF MS.

Search “ziptip” on www.emdmillipore.com for details.

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Ordering InformationTo 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/pkIncludes reagent kit (resin & buffers)

Reagent Kit Only

MWCO

3,000 10,000 30,000 50,000 100,000

Amicon® Pro Affinity Concentration Kit Ni-NTA ACR5000NT ACK5003NT ACK5010NT ACK5030NT ACK5050NT ACK5100NT

Amicon® Pro Affinity Concentration Kit Protein A ACR5000PA ACK5003PA ACK5010PA ACK5030PA ACK5050PA ACK5100PA

Amicon® Pro Affinity Concentration Kit Protein G ACR5000PG ACK5003PG ACK5010PG ACK5030PG ACK5050PG ACK5100PG

Amicon® Pro Affinity Concentration Kit GST ACR5000GS ACK5003GS ACK5010GS ACK5030GS ACK5050GS ACK5100GS

Amicon® Pro purification system – No Reagents Included 

MWCO

3,000 10,000 30,000 50,000 100,000

Amicon® Pro Purification System Trial Pack 2/pk ACS500302 ACS501002 ACS503002 ACS505002 ACS510002

Amicon® Pro Purification System 12/pk ACS500312 ACS501012 ACS503012 ACS505012 ACS510012

Amicon® Pro Purification System 24/pk ACS500324 ACS501024 ACS503024 ACS505024 ACS510024

• Ni-NTA Resins• HIS•Bind™ Resins• GST Resins• Strep-Tactin Agarose

Related Products

• S-tag Agarose• T7 tag Agarose• Cleavage Enzymes - Factor

Xa, Thrombin, Enterokinase

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.

To Place an Order or ReceiveTechnical AssistanceIn 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

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Only one device required for sample prep workflow, including:• Purification• Concentration•Desalting / buffer exchange

Superior sample recovery•No need to transfer• Better protein binding

with packed agarose resin compared to batch mixing

Ergonomic, less pipetting•No need to transfer

Simultaneous concentration capability• Elution without dilution• Consistent recovery• Saves time

Large reservoir saves time•Holds wide range of volumes• Reduces spin steps• Reservoir cap can be labeled

to reduce time and confusion

Gentle and efficient buffer exchange• >99.99% (1000-fold)

desalting in a single spin•Maintains protein activity

Broad reagent compatibility • Agarose resin kits optimized

for the Amicon® Pro device • Compatible with other resins

>10 μm in diameter• Chemically compatible with

most common buffers

Components, features and benefits of the Amicon® Pro purification system

Exchange Device CapEasy-to-label cap can be used in place of collection tube cap in centrifuge

Amicon® Ultra 0.5 mL Filter Collection TubeReverse spin sample collection

Easy, efficient, reproducible

Collection Tube Cap

Exchange Device Larger size holds larger wash volumes

Unique tip fits into Amicon Ultra® 0.5 mL filter

Enables single spin buffer exchange

Amicon® Ultra 0.5 mL Filter Consistent concentration with the Amicon Ultra® 0.5 mL filter

Provides elution without dilution

Enables gentle diafiltration buffer exchange

Collection Tube Large volume 50 mL tube

No need to empty between spins