Agilent Human 14
Multiple Affinity
Removal System Spin
Cartridges for the
Depletion ofHigh-AbundantProteins from Human
Proteomic Samples
Instructions
Second edition
October 2008

General Information
Introduction
The Agilent Human 14 Multiple Affinity Removal
System comprises a family of immunodepletion
products based on affinity interactions and optimized buffers for sample loading, washing, eluting,and regenerating. This spin cartridge is specificallydesigned to remove 14 high-abundant proteins fromhuman biological fluids such as plasma, serum, andcerebral spinal fluid (CSF). This technology enables
removal of albumin, IgG, antitrypsin, IgA, transferrin,
haptoglobin, fibrinogen, alpha2-macroglobulin,
alpha1-acid glycoprotein, IgM, apolipoprotein AI,
apolipoprotein AII, complement C3, and transthyretin with a single device. The targeted high-abundant
proteins are simultaneously removed when crude
biological samples are passed through the cartridge.
Selective immunodepletion provides an enrichedpool of low-abundant proteins for downstream proteomics analysis.
Specific removal of 14 high-abundant proteinsdepletes approximately 94% of total protein massfrom human serum/plasma. The low-abundant proteins in the flow-through fractions can be studied.
Removal of high-abundant proteins enables
improved resolution and dynamic range for
one-dimensional gel electrophoresis (1DGE),
two-dimensional gel electrophoresis (2DGE), andliquid chromatography/mass spectrometry
Instructions2

(LC/MS). The collected flow-through fractions mayneed to be concentrated dependent upon the downstream applications.

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Human 14 Multiple Affinity Removal
SystemThe Agilent Human 14 Multiple Affinity Removal
System is specially designed to allow for close studyof low-abundant proteins present in flow-throughfractions (see Figure 1).

H H
L H L
H L LHL H
H LH L H

Apply Crude Human
Serum/Plasma

Human Proteins Depleted:
Albumin, IgG, Antitrypsin,
IgA, Transferrin, Haptoglobin,
Fibrinogen, Alpha2-Macroglobulin,
Alpha1-Acid Glycoprotein, IgM,
Apolipoprotein AI, Apolipoprotein AII,
Complement C3, Transthyretin

L L
L LL LL

Fraction #1
Low-AbundantProteins

HH
H H
H
H HH

Fraction #2
High-AbundantProteins

Figure 1 The Multiple Affinity Removal System.
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Product Description
The Agilent Human 14 Multiple Affinity Removal
System Spin Cartridge and its accessories areshown in Table 1.

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Table 1 Agilent Human 14 Multiple Affinity
Removal System Spin Cartridge andStarter Reagent Kit
Product no. Product name Product description
5188-6560 0.45-mL affinity, Removes human albumin,
spin cartridge, IgG, antitrypsin, IgA, trans1 eachferrin, haptoglobin, fibrinogen, alpha2-macroglobulin, alpha1-acid glycoprotein, IgM, apolipoprotein
AI, apolipoprotein AII,
complement C3, and transthyretin
5185-5987 Buffer A, 1 L
Ready-to-use, optimized
buffer for loading, washing, and equilibrating cartridge
5185-5988 Buffer B, 1 L
Ready-to-use, optimized
buffer for elution of boundproteins from cartridge
5185-5990 Spin filters
For sample cleanup before
0.22 µm,
loading cartridge
1 pack of 25
5185-5991 Protein spin
For concentrating
concentrators, flow-through fractions
5kDa MWCO,
4 mL, 1 packof 25
5188-5249 Luer-Lock
Allows attachment ofadapters,
Luer-Lock syringes to spin
1 pack of 2
cartridge

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Product no. Product name Product description
5188-5250 5-mL plastic
For washing, eluting, andLuer-Lockre-equilibrating buffers
syringes,through spin cartridge
1 pack of 25188-5251 1.5-mL screwtop Eppendorf-style tubes
microtube,
used for collecting frac1 pack of 100 tions from spin cartridge
5188-5252 Spin cartridge Extra caps and plugs forscrew caps
sealing the top and bottom
and plugs,
of affinity spin cartridges
1 pack of 6 each
5188-5253 Teflon LuerFor transferring solutions
Lock needles, with Luer-Lock Syringes
1 pack of 105188-5254 Starter Reagent Kit for Spin Cartridges*
Buffer A: 1 L
Buffer B: 1 L
Spin filters 0.22 µm: 2 packs of 25
Protein concentrators: 1 pack of 25
Luer-Lock adapters: 1 pack of 2
5-mL plastic Luer-Lock syringes: 1 pack of 2
1.5-mL microtubes: 6 packs of 100
Spin cartridge extra caps and plugs, 1 packof 6 each
Teflon Luer-Lock needles, 1 pack of 10* Under normal conditions, the kit should last for approximately 200 spin cartridge uses.

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N O TE

Instructions

For higher capacity Multiple Affinity Removal
System Devices, and if automated immunodepletion is needed, refer to Human 14 Multiple Affinity Removal System Columns (part number
5188-6557 - 4.6 x 50 mm, 5188-6558 - 4.6 x 100
mm, 5188-6559 - 10 x 100 mm) for use with HPLC
instrumentation.
See www.agilent.com/chem/bioreagents formore information and links to mouse protein
removal devices.

8Full Protocol for Human 14 Multiple
Affinity Removal System Spin Cartridge(Cartridge capacity: 8–10 µL human serum/plasma*)During use, never let the spin cartridge frits orresin bed run dry. If this happens, see Recommendations section for cartridge rewetting procedure.

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Additional Materials Required• Microcentrifuge with adjustable centrifugal force(capable of spinning at 100 x g) and timer such as
the Eppendorf Model 5415D• 50-mL vessels (for example, polypropylene tubes)
to hold working quantities of Buffers A and B during procedure• Adjustable pipettes for delivering up to 400-µL aliquots* Capacity testing was done with pooled normal plasma
from Rockland Immunochemicals (D519-04).
Consult certificate of analysis to verify your capacity.
It is the user's responsibility to adjust loading volume tocompensate for the fact that the concentrations of somehigh-abundant proteins can vary widely depending on thesample origin (source). Several serum/plasma proteinssuch as alpha1-antitrypsin, haptoglobin, fibrinogen, IgG,
and alpha1-acid glycoprotein rise several folds in
response to stress, infection, inflammation, or tissue
necrosis and are known as acute phase reactants (Henry,
J.B. 1996 - Clinical Diagnosis and Management by Laboratory Methods).

Instructions

9• Transfer pipettes• 1.5-mL screw-top microtubes/Eppendorf-styletubes (collection tubes, part number 5188-5251)• Luer-Lock adapters (part number 5188-5249)• 5-mL plastic Luer-Lock syringes (part number
5188-5250)• Buffer A, 1 L (part number 5185-5987)• Buffer B, 1 L (part number 5185-5988)

Material Preparation• Fill two 50-mL vessels with appropriate amountsof Buffers A and B to use throughout the procedure for the specific number of samples you wish
to process (approximately 5-mL Buffer A and
2-mL Buffer B per each 8–10 µL serum/plasma sample).
• Label two 5-mL Luer-Lock syringes with "A" and"B" for using later in the procedure during the
cartridge elution and re-equilibration steps.
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Instructions

Remove Agilent Human 14 Multiple Affinity
Removal System Spin Cartridge from refrigerator
and allow cartridge to equilibrate to room temperature before use.

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Procedure
1 Dilute and filter sample. Prepare the sample bydiluting 8–10 µL of human serum/plasma* withBuffer A to a final volume of 200 µL. For example:
if the recommended cartridge loading capacity
on the certificate is 10 µL of serum/plasma,dilute 10 µL of serum/plasma with 190-µL
Buffer A† to a final volume of 200 µL.
If you plan to perform several successive runs on
the cartridge, increase amount of diluted sample
accordingly.
Filter diluted samples through a 0.22-µm spin filter (part number 5185-5990) to prevent clogging
of spin cartridge frits.
2 Prepare spin cartridge. Remove cartridge cap
and plug. Attach Luer-Lock adapter to spin cartridge, draw 4 mL of Buffer A into syringe andthen attach it to Luer-Lock on spin cartridge.
Dispense Buffer A through spin cartridge to prepare resin and to remove any trapped air bubbles. With a transfer pipette, remove excess
Buffer A from top of the spin cartridge.

* Protocol may be applied to other human biological fluidswith necessary adjustments in sample volume based onalbumin concentration.
† Addition of protease inhibitors in Buffer A for sample
dilution helps prevent protein degradation.

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3 Apply sample. Remove the Luer-Lock adapter
and place spin cartridge in a screw-top collection
tube and label it "Flow-through 1" or "F1". Add
200 µL of diluted serum/plasma sample to spin
cartridge and centrifuge for 1.0 minute at 100 × g(or lowest possible setting on centrifuge; seenote below). Cap the spin cartridge loosely or
leave open during centrifugation so the sample
is able to flow. Collect the flow-through fractionin the collection tube F1. The resin bed and frits
should remain moist, not dry, after centrifugation.
If centrifuge cannot be programmed to 100 x g,then cartridge capacity may be different for use;the optimum results for depletion are obtained
when the flow rate is controlled to < 0.2 mL/min.

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4 Incubate. Remove spin cartridge from centrifuge
and allow to sit for 5 minutes at room temperature.
5 Wash and collect flow-through fraction F1.
Add 400 µL of Buffer A to the top of the resin bed
and centrifuge for 2.5 minutes at 100 × g. Collectthe flow-through fraction into the same F1 collection tube.
6 Wash and collect additional flow-through fraction F2. Place spin cartridge into a new collection tube labeled "Flow-through 2" or "F2". Add
400 µL of Buffer A to the top of the resin bed andInstructions

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centrifuge for 2.5 minutes at 100 × g. Collect theflow-through fraction into the F2 collection tube.
7 Prepare for elution. Remove the spin cartridgefrom the F2 collection tube and attach Luer-Lockadapter to the cartridge top.
8 Elute bound fraction. Fill a 5-mL plastic
Luer-Lock syringe (labeled "B") with 2.5 mL of
Buffer B and attach to the spin cartridge via theLuer-Lock adapter. Elute bound high-abundant
proteins into a new collection tube by slowly
pushing Buffer B through the spin cartridge.
Save the bound fraction for analysis if desired, ordiscard it. Do not push air through the spin cartridge and do not allow the resin bed or frits torun dry.
If the meniscus of Buffer B does not reach the
top frit after depressing the syringe plunger completely, remove syringe and draw the plunger
back to the 1-mL mark with air and reattach tothe cartridge. Use the air in the syringe as positive pressure to push Buffer B through until themeniscus of Buffer B reaches the top frit.

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9 Re-equilibrate. Remove Buffer B syringe andattach a 5-mL syringe (labeled "A") containing
5 mL of Buffer A to the spin cartridge. Re-equilibrate the spin cartridge by slowly pushing
Buffer A through the resin bed. Do not allow theresin bed or frits to run dry by leaving a small aliquot of buffer on the top of the frit. The spin
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cartridge is ready for the next sample. For storage, leave the resin bed wet with Buffer A, andleave a layer of Buffer A above the top frit. Recap
both ends of the spin cartridge tightly.
Be careful when placing the plug in lower end of
the spin cartridge; it must not displace or puncture the lower frit. Store spin cartridge in Buffer
A in a refrigerator at 2–8 °C (35–46 °F). DO NOTFREEZE THE SPIN CARTRIDGE.

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10 Analyze. Analyze separately or combine
flow-through fractions F1 and F2 containing the
low-abundant proteins. For 1D-SDS-PAGE, an aliquot of the flow-through fraction may be useddirectly. For IEF, 2DGE, and MS analysis of theflow-through fraction, it is necessary to buffer
exchange/desalt to an appropriate buffer. The
5KDa MWCO spin concentrators (part number
5185-5991) may be used for buffer exchange andconcentration. Alternatively, for automateddesalting and concentration, the Agilent
mRP-C18 column (part number 5188-5231) may
be used according to published methods (Agilent
Technologies, publication 5989-2506EN).

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Instructions

For maximum recovery of the flow-throughprotein combine and concentrate fractions F1
and F2.

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Recommendations• Sample dilution
It is not recommended to load crude serum or
plasma directly onto the spin cartridge. Follow
instructions for serum/plasma dilution. Additionof protease inhibitors in Buffer A for sample dilution helps prevent protein degradation.
• Sample cleanup
Human serum or plasma may contain particulatematerials that can be removed by a quick spinusing a 0.22-µm spin filter (part number
5185-5990).
• Sample concentration
For further downstream proteomic analysis(SDS-PAGE or LC/MS), combine flow-throughfractions F1 and F2 and concentrate the samples.
Spin concentrators with 5 KDa MWCO (part number 5185-5991) can be used to concentrate proteins before analysis. Alternatively, for automateddesalting and concentration, the Agilent
mRP-C18 column (part number 5188-5231) may
be used according to published methods (Agilent
Technologies, publication 5989-2506EN).
• Bound fraction analysis
If you will be analyzing the bound fraction, perform a buffer exchange to phosphate-bufferedsaline (PBS) or to another buffer compatible withyour analysis. Buffer B contains compounds that
may interfere with some protein assays.

Instructions15

• Cartridge rewetting
If the resin bed becomes dry during cartridge centrifugation or syringe elution, attach a syringewith Buffer A to the spin cartridge via theLuer-Lock adapter and re-equilibrate the cartridgeby passing Buffer A through it. This should notaffect the spin cartridge performance.
• Spin cartridge performance
Human 14 Multiple Affinity Removal System Spin
Cartridges should perform reproducibly for morethan 200 runs under proper conditions. Buffers A
and B are optimized to support cartridge performance and longevity. Agilent does not guarantee
spin cartridge performance if other buffers areused. Do not expose cartridges to organic solvents (like alcohols, acetonitrile, etc.), strong oxidizers, acids, or reducing agents, and otherprotein denaturing agents.
• Cartridge storage
After equilibrating Multiple Affinity Removal System Spin Cartridges with Buffer A, always store
them in a refrigerator at 2–8 °C (35–46 °F) when
not in use to minimize loss in cartridge capacity.
• Lyophilization of flow-through fractions
Buffer exchange to a volatile buffer (for example,
ammonium bicarbonate) is recommended prior tolyophilization due to high salt concentration in
Buffer A.

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Troubleshooting
Problem
No flow

Cause
Solution
The spin cartridge Remove or loosen
may be capped too cap during centrifutightly during cen- gation.
trifugation.
Bubble under resin Rewet with Buffer Aor frits.
(see Recommendations - Cartridge
rewetting).
Incomplete Centrifugation para- Adjust centrifuge
flowmeters need to be force and time toadjustedachieve≤ 0.2-mL/min flow
rates through spin
cartridge during
steps 3 through 5 ofprocedure.
No proteins Buffers A and B
Re-equilibrate spinin boundreversed
cartridge withfraction
Buffer A (step 8) andstart over with correct buffer sequence.

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Problem
Cause
Solution
BreakExceeding cartridge Reduce
through of serum/plasma
serum/plasma loadhighcapacity
per sample.
abundant
proteins in Serum/plasma pro- Reduce
flow-through tein levels may be serum/plasma load
fractions F1 unusually highper sample.
and F2
Flow rate through Reduce centrifugacartridge during
tion force and/or
sample loading too time during sample
highloading to not exceed
0.2 mL/min flow rate.

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Cartridge Specifications
Part number 5188-6560

Cartridge body material
Frit materials
Cartridge capacity*
Recommended centrifugal
force
Operating temperature
Cartridge packing material
Immobilized ligands

Shipping solution
Shipping temperature
Storage temperature

0.45-mL Multiple Affinity
Removal System Cartridge,
1 eachPolypropylene
Polyethylene with 10-µm
pore size
8–10 µL humanserum/plasma
100 x g
18–25 °C
Affinity resin
Affinity ligands to humanalbumin, IgG, antitrypsin,
IgA, transferrin, haptoglobin,
fibrinogen, alpha2-macroglobulin, alpha1-acid glycoprotein, IgM, apolipoprotein
AI, apolipoprotein AII, complement C3, and transthyretin
Buffer A with 0.02% sodium
azide
2–8 °C (35–46 °F)
2–8 °C (35–46 °F)* For exact cartridge capacity, consult your cartridge certificate of analysis.

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Do not expose cartridges to organic solvents(like alcohols, acetonitrile, etc.), strong oxidizers, acids, or reducing agents, and other protein denaturing agents.

CAU T ION

For RESEARCH USE ONLY. This product is
NOT TO BE USED AS AN IN-VITRODIAGNOSTIC.

WARN IN G

Storage: Store the affinity cartridge at 2–8 °C(35–46 °F) upon receiving and when not in use. DO
NOT FREEZE THE CARTRIDGE! Store cartridge wetted with Buffer A and with the end-caps tightly
sealed.

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Safety Issues
When preparing biological samples using Agilent
Human 14 Multiple Affinity Removal System Spin
Cartridges, follow general guidelines for handling
biological materials and wear protective eyewear
and gloves.

Related Agilent Products
Other related Agilent products include the following:
5188-6557 Agilent Multiple Affinity Removal System
Column Hu-14, 4.6 X 50 mm, LC columnthat depletes 14 high-abundant proteins(albumin, IgG, IgA, transferrin, haptoglobin, antitrypsin, fibrinogen, alpha2-macroglobulin, alpha1-acid glycoprotein, IgM,
apolipoprotein AI, apolipoprotein AII, complement C3, and transthyretin ) from humanserum/plasma samples, 20 µL serum/plasma capacity per injection

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5188-6558 Agilent Multiple Affinity Removal System
Column Hu-14, 4.6 X 100 mm, LC columnthat depletes 14 high-abundant proteins(albumin, IgG, IgA, transferrin, haptoglobin, antitrypsin, fibrinogen, alpha2-macroglobulin, alpha1-acid glycoprotein, IgM,
apolipoprotein AI, apolipoprotein AII, comple- ment C3, and transthyretin) fromhuman serum/plasma samples, 40 µL
serum/plasma capacity per injection
5188-6559 Agilent Multiple Affinity Removal System
Column Hu-14, 10 X 100 mm, LC columnthat depletes 14 high-abundant proteins(albumin, IgG, IgA, transferrin, haptoglobin, antitrypsin, fibrinogen, alpha2-macroglobulin, alpha1-acid glycoprotein, IgM,
apolipoprotein AI, apolipoprotein AII,
complement C3, and transthyretin) fromhuman serum/plasma samples, 250 µL
serum/plasma capacity per injection
5188-6409 Agilent High Capacity Multiple Affinity
Removal System Column Hu-7HC,
4.6 X 50 mm, LC column that depletes
seven high-abundant proteins (albumin,
IgG, IgA, transferrin, haptoglobin, antitrypsin, and fibrinogen) from humanserum/plasma samples, 30–35 µL
serum/plasma capacity per injection

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5188-6410 Agilent High Capacity Multiple Affinity
Removal System Column Hu-7HC,
4.6 X 100 mm, LC column that depletes
seven high-abundant proteins (albumin,
IgG, IgA, transferrin, haptoglobin, antitrypsin, and fibrinogen) from humanserum/plasma samples, 60–70 µL
serum/plasma capacity per injection
5188-6411 Agilent High Capacity Multiple Affinity
Removal System Column Hu-7HC,
10 X 100 mm, LC column that depletes
seven high-abundant proteins (albumin,
IgG, IgA, transferrin, haptoglobin, antitrypsin, and fibrinogen) from humanserum/plasma samples, 250–300 µL
serum/plasma capacity per injection
5188-6408 Agilent High Capacity Multiple Affinity
Removal System Spin Cartridge Hu-7HC,
spin cartridge that depletes sevenhigh-abundant proteins (albumin, IgG, IgA,
transferrin, haptoglobin, antitrypsin, andfibrinogen) from human serum/plasma
samples, 12–14 µL serum/plasma capacity
per use5188-5332 Agilent High Capacity Multiple Affinity
Removal System Column Hu-6HC,
4.6 X 50 mm, LC column that depletes six
high-abundant proteins (albumin, IgG, IgA,
transferrin, haptoglobin, and antitrypsin)from human serum/plasma samples,
30–40 µL serum/plasma capacity per injection

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5188-5333 Agilent High Capacity Multiple Affinity
Removal System Column Hu-6HC,
4.6 X 100 mm, LC column that depletes six
high-abundant proteins (albumin, IgG, IgA,
transferrin, haptoglobin, and antitrypsin)from human serum/plasma samples,
60–80 µL serum/plasma capacity per injection
5188-5336 Agilent High Capacity Multiple Affinity
Removal System Column Hu-6HC,
10 X 100 mm, LC column that depletes six
high-abundant proteins (albumin, IgG, IgA,
transferrin, haptoglobin, and antitrypsin)from human serum/plasma samples,
300–325 µL serum/plasma capacity per
injection
5188-5341 Agilent High Capacity Multiple Affinity
Removal System Spin Cartridge Hu-6HC,
spin cartridge that depletes six high-abundant proteins (albumin, IgG, IgA, transferrin, haptoglobin, and antitrypsin) fromhuman serum/plasma samples, 14–16 µL
serum/plasma capacity per use
5185-5984 Multiple Affinity Removal System Column
Hu-6, 4.6 × 50 mm, LC column that
depletes six high-abundant proteins (albumin, IgG, IgA, transferrin, haptoglobin, andantitrypsin) from human serum/plasma
samples, 15–20 µL serum/plasma capacity
per injection

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5185-5985 Multiple Affinity Removal System Column
Hu-6, 4.6 × 100 mm, LC column that
depletes six high-abundant proteins (albumin, IgG, IgA, transferrin, haptoglobin, andantitrypsin) from human serum/plasma
samples, 30–40 µL serum/plasma capacity
per injection
5188-2714 Multiple Affinity Removal System Column
Hu-6, 10 × 100 mm, LC column that
depletes six high-abundant proteins (albumin, IgG, IgA, transferrin, haptoglobin, andantitrypsin) from human serum/plasma
samples, 140–190 µL serum/plasma
capacity per injection
5188-5230 Multiple Affinity Removal System Spin
Cartridge Hu-6, spin cartridge that
depletes six high-abundant proteins (albumin, IgG, IgA, transferrin, haptoglobin, andantitryp- sin) from human serum/plasma
samples, 7–10 µL serum/plasma capacity
per use5188-5217 Multiple Affinity Removal System Column
Ms-3, 4.6 × 50 mm, LC column that
depletes three high-abundant proteins(albumin, IgG, and transferrin) from mouseserum/plasma samples, 37–50 µL
serum/plasma capacity per injection
5188-5218 Multiple Affinity Removal System Column
Ms-3, 4.6 × 100 mm, LC column that
depletes three high-abundant proteins(albumin, IgG, and transferrin) from mouseserum/plasma, 75–100 µL serum/plasma
capacity per injection

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5188-5289 Multiple Affinity Removal System Spin
Cartridge Ms-3, spin cartridge that
depletes three high-abundant proteins(albumin, IgG, and transferrin) from mouseserum/plasma samples, 25–30 µL
serum/plasma capacity per use
5185-5986 Multiple Affinity Removal System Reagent
Kit, starter reagent kit containing buffers,
spin filters, and spin concentrators for use
with Multiple Affinity Removal System LC
Columns
5188-5254 Starter Reagent Kit for Spin Cartridges*
Buffer A: 1 L
Buffer B: 1 L
Spin filters 0.22 µm: 2 packs of 25
Protein concentrators: 1 pack of 25
Luer-Lock adapters: 1 pack of 2
5-mL plastic Luer-Lock syringes: 1 pack of 2
1.5-mL microtubes: 6 packs of 100
Spin cartridge extra caps and plugs, 1 packof 6 each
Teflon Luer-Lock needles, 1 pack of 105188-5231 mRP-C18 High Recovery Protein Fractionation and Desalting Column, seewww.agilent.com/chem/bioreagents formore details* Under normal conditions, the kit should last for approximately 200 spin cartridge uses.

For more information or technical assistance, pleasecall toll free: 1-800-227-9770 or visit our Web site at:www.agilent.com/chem/bioreagents.
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Instructions

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©Agilent Technologies, Inc. 2008
Agilent shall not be liable for errors
contained herein or for incidental orconsequential damages in connection
with the furnishing, performance, or
use of this material.
Information, descriptions, and specifications in this publication are subject
to change without notice.
Printed in the USA
Second edition
October 2008
5969-1597