5188-5290.book Page 1 Thursday, October 30, 2008 5:22 PMAgilent Multiple
Affinity Removal Spin
Cartridges for theDepletion ofHigh-AbundantProteins from Mouse
Proteomic SamplesInstructions
Third edition
October 2008

5188-5290.book Page 2 Thursday, October 30, 2008 5:22 PM

General Information
Introduction
The Agilent Multiple Affinity Removal System comprises a family of immunodepletion products basedon antibody-antigen interactions and optimized buffers for sample loading, washing, eluting, and regenerating. This spin cartridge is specifically designedto remove three high-abundant proteins from mouse
biological fluids such as serum and plasma. This
technology enables removal of albumin, IgG, andtransferrin with a single device. The targeted
high-abundant proteins are simultaneously removedby the immobilized antibodies when crude biologicalsamples are passed through the cartridge. Selective
immunodepletion provides an enriched pool of
low-abundant proteins for downstream proteomics
analysis. Removal of high-abundant proteinsenables improved resolution and dynamic range for
one-dimensional gel electrophoresis (1DGE),
two-dimensional gel electrophoresis (2DGE), andliquid chromatography/mass spectrometry(LC/MS). The collected flow-through fractions mayneed to be concentrated dependent upon the downstream applications.

Instructions

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Multiple Affinity Removal System
The Agilent Multiple Affinity Removal System is
specially designed to allow for close study of
low-abundant proteins present in flow-through fractions (see Figure 1).

H H
L H L
H L LHL H
H LH L H
Crude Mouse Serum

L L
L LL LL
Low-Abundant Proteins
Free from Interferences

HHigh-Abundant Proteins(Albumin, IgG, and Transferrin)

L

Low-Abundant Proteins(Biomarkers for disease and drug targets)

Figure 1 The Multiple Affinity Removal System.

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Product Description
The Multiple Affinity Removal Spin Cartridge and its
accessories are shown in Table 1.
Table 1 Multiple Affinity Removal Spin Cartridge
and Starter Reagent Kit
Product no. Product name Product description
5188-5289 0.45-mL affinity, Removes mouse albumin,
spin cartridge, IgG, and transferrin
1 each
5185-5987 Buffer A, 1 LReady-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, 1 pack loading cartridgeof 255185-5991 Protein spin
For concentrating
concentrators, flow-through fractions
5k Da MWCO,
4 mL, 1 packof 25
5188-5249 Luer-Lock
Allows attachment ofadapters, pack Luer-Lock syringes to spinof 2cartridge

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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 Screw- Eppendorf-style tubes
top 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 forapproximately 200 spin cartridge uses.

Instructions

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N O TEFor higher capacity Multiple Affinity RemovalDevices, and if automated immunodepletion isneeded, refer to Multiple Affinity Removal Columns(part number 5188-5217, 4.6 × 50 mm and 5188-5218,
4.6 × 100 mm columns for mouse serum) for use
with HPLC instrumentation.

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Full Protocol for 0.45-mL Multiple Affinity
Removal Spin Cartridge(Cartridge capacity: 25-30 µL mouse serum*)N O TEDuring use, never let the spin cartridge frits or resin
bed run dry. If this happens, see “Recommendations” section for a cartridge rewetting procedure.
* Consult spin cartridge certificate of analysis to verify
your cartridge capacity. Capacity was determinedusing pooled Swiss Webster mouse serum. Mouse
serum protein concentrations can vary for otherstrains and your sample size should be adjusted
accordingly.

Additional Materials Required• Microcentrifuge with adjustable centrifugal force(capable of spinning at 100 × g) and timer such as
the Eppendorf Model 5415D• 50-mL vessels (for example, polypropylene tubes)
to hold small quantities of Buffers A and B during
procedure• Adjustable pipettes for delivering up to 400-µL
aliquots• Transfer pipettes• 1.5-mL screw-top Microtubes/Eppendorf-styletubes (collection tubes, part number 5188-5251)• Luer-Lock adapters (part number 5188-5249)(continued on next page)
Instructions

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5188-5290.book Page 8 Thursday, October 30, 2008 5:22 PM• 5-mL plastic Luer-Lock syringes (part number5188-5250)• 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 your specific number of samples you
wish to process (approximately 5-mL Buffer A
and 2-mL Buffer B per each 25-30 µL serumsample).
• Label two 5-mL Luer-Lock syringes with “A” and“B” for using later in the procedure during thecartridge elution and re-equilibration steps.

N O TE

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

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5188-5290.book Page 9 Thursday, October 30, 2008 5:22 PMProcedure
1 Dilute and filter sample. Prepare your sample bydiluting 25-30 µL of mouse serum* (consult cartridge certificate for actual serum capacity) withBuffer A† to a final volume of 200 µL. For example: if the recommended cartridge loading capacity on the certificate is 30 µL of serum, dilute
30 µL of serum with 170-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 sampleaccordingly.
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 capand 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 any excess Buffer A from top of the spin cartridge.

* Protocol may be applied to other mouse biologicalfluids like plasma.
† Addition of protease inhibitors in Buffer A for sample
dilution helps prevent protein degradation.

Instructions

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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 sample to top of resin
bed and centrifuge for 1.5 minutes at 100 × g (orlowest possible setting on centrifuge - See notebelow). Cap the spin cartridge loosely or leave
open during centrifugation so the sample is ableto flow. Collect the flow-through fraction in thecollection tube F1. The resin bed and frits should
remain moist, not dry after centrifugation.

N O TEIf centrifuge cannot be programmed to 100 × g, thencartridge capacity may be different for use; the optimum results for depletion are obtained when theflow rate is controlled to 0.2 mL/min.
4 Wash and collect flow-through fraction F1. Add
400 µL of Buffer A to the top of the resin bed andcentrifuge for 2.5 minutes at 100 × g. Collect theflow-through fraction into the same F1 collection
tube.
5 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 andcentrifuge for 2.5 minutes at 100 × g. Collect theflow-through fraction into the F2 collection tube.
6 Prepare for elution. Remove the spin cartridgefrom the F2 collection tube and attach Luer-Lockadapter to the cartridge top.

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7 Elute bound fraction. Fill a 5-mL plastic
Luer-Lock syringe (labeled “B”) with 2 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.

N O TE

If the meniscus of Buffer B does not reach the top
frit after depressing the syringe plunger completely,remove syringe and draw plunger back to the 1-mL
mark with air and re-attach to the cartridge. Use theair in the syringe as positive pressure to push Buffer
B through until the meniscus of Buffer B reaches thetop frit.
8 Re-equilibrate. Remove Buffer B syringe andattach a 5-mL syringe (labeled “A”) containing
4 mL of Buffer A to the spin cartridge.
Re-equilibrate 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 cartridge is ready for the next sample. For storage,leave the resin bed wet with Buffer A, and leavea layer of Buffer A above the top frit. Recap bothends of the spin cartridge tightly.

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N O TEBe careful when placing plug in lower end that it
does not displace or puncture the lower frit. Storespin cartridge in Buffer A in a refrigerator at 2-8 °C(35-46 °F). DO NOT FREEZE THE SPIN CARTRIDGE.
9 Analyze. Analyze separately or combine
flow-through fractions F1 and F2 containing thelow-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 number5185-5991) 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 (AgilentTechnologies, publication 5989-2506EN). For
1D-SDS-PAGE, the flow-through fraction may beused directly. For IEF, 2DGE, and MS analysis of
the flow-through fraction, buffer exchange to an
appropriate buffer is necessary.

N O TEFor maximum recovery of the flow-through protein
combine and concentrate fractions F1 and F2.

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Recommendations• Sample dilution
It is not recommended to load crude serum
directly onto the spin cartridge. Follow instructions for serum dilution. Addition of proteaseinhibitors in Buffer A for sample dilution helps
prevent protein degradation.
• Sample cleanup
Mouse serum may contain particulate materials
that can be removed by a quick spin using a
0.22-µm spin filter (part number 5185-5990).
• Sample concentration
For further down-stream proteomic analysis(SDS-PAGE or LC/MS), combine flow-throughfractions F1 and F2 and concentrate the samples.
Spin concentrators with 5 KDa MWCO (partnumber 5185-5991) can be used to concentrate
proteins before analysis.
• Bound fraction analysis
If you prefer to analyze 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.

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5188-5290.book Page 14 Thursday, October 30, 2008 5:22 PM• 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
Multiple Affinity Removal Spin Cartridges should
perform reproducibly for at least 200 runs underproper conditions. Buffers A and B are optimized
to support cartridge performance and longevity.
We cannot guarantee spin cartridge performance
if other buffers are used. Do not expose cartridges to organic solvents (like alcohols, acetonitrile, etc.), strong oxidizers, acids, or reducing
agents, and other protein denaturing agents.
• Cartridge storage
Always store Multiple Affinity Removal Spin Cartridges after equilibrating with Buffer A in arefrigerator at 2-8 °C (35-46 °F) when not in use
to minimize loss in cartridge capacity.
• Lyophylization of flow-through fractions
Buffer exchange to a volatile buffer (for example,ammonium bicarbonate) is recommended prior tolyophylization due to high salt concentration in
Buffer A.

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Troubleshooting
Problem
No flowCauseSolutionThe spin cartridge Remove or loosen
may be capped too cap during
tightly during
centrifugation.
centrifugation.
Bubble underresin or frits.

Incomplete See No flow.
flowCentrifuge
parameters need
to be adjusted

No proteins Buffers A and B
in boundreversed
fraction

Instructions

Rewet with Buffer A(see “Recommendations - Cartridgerewetting”).
-Adjust centrifuge
force and time toachieve ≤0.2-mL/minflow rates throughspin cartridge during
step 3 through step 5of procedure.
Re-equilibrate spin
cartridge with Buffer
A (step 8) and startover with correct buffer sequence.

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Problem
Breakthrough ofhighabundant
proteins
in flowthroughfractions
F1 and F2

Instructions

Cause
Solution
Exceeding cartridge Reduce serum load
serum capacity
per sample.
Serum protein
levels may be
unusually highReduce serum load
per sample.

Flow rate throughspin cartridge
during sample
loading too highReduce centrifugation force and/ortime during sample
loading to not exceed
0.2 mL/min flow rate.

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Cartridge Specifications
Parameter
Part number 5188-5289

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

Shipping solution
Shipping temperature
Storage temperature

Description
0.45 mL Multiple Affinity
Removal Spin Cartridge
1 eachPolypropylene
Polyethylene with 10-µm
pore size
25-30 µL mouse serum
100 × g18-25 °C
Antibody-modified resin
Affinity-purified polyclonal
antibodies to mouse albumin, IgG, and transferrin
Buffer A with 0.02% sodiumazide
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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Safety Issues
When preparing biological samples using Multiple
Affinity Removal Spin Cartridges, follow general
guidelines for handling biological materials andwear protective eyewear and gloves.

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Related Agilent Products
Part number Description
5188-5230 Multiple Affinity Removal Spin Cartridge
Hu-6, 0.45 mL, spin cartridge that depletes
six high-abundant proteins (albumin, IgG,IgA, transferrin, haptoglobin, andantitrypsin) from human serum samples,
7-10 µL serum capacity per use
5185-5984 Multiple Affinity Removal Column Hu-6,
4.6 × 50 mm, LC column that depletes six
high-abundant proteins from humanserum samples, 15-20 µL serum capacity
per injection
5185-5985 Multiple Affinity Removal Column Hu-6,
4.6 × 100 mm, LC column with 30-40 µL
human serum capacity per injection
5188-5217 Multiple Affinity Removal Column Ms-3,4.6 × 50 mm, LC column that depletes
three high-abundant proteins from mouseserum samples, 37-50 µL serum capacity
per injection
5188-5218 Multiple Affinity Removal Column Ms-3,
4.6 × 100 mm, LC column with 75-100 µL
mouse serum capacity per injection
5185-5986 Multiple Affinity Removal System
Reagent Kit, starter reagent kit containing
buffers, spin filters, and spin concentrators for use with Multiple Affinity Removal
LC Columns

Instructions

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5188-5290.book Page 20 Thursday, October 30, 2008 5:22 PMFor more information or technical assistance, pleasecall toll free: 1-800-227-9770 or visit our website at:www.agilent.com/chem/bioreagents.

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

CAU T IONWARN IN G

For RESEARCH USE ONLY - this product is NOT
TO BE USED AS AN IN-VITRO DIAGNOSTIC.
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.

Instructions

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5188-5290.book Page 22 Thursday, October 30, 2008 5:22 PM©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
Third edition
October 2008
5188-5290