1 Dilute and filter sample

2 Prepare spin cartridge

3 Apply sample

4 Wash and collect

5 Wash and collectflow-through F1

flow-through F2

F1

F1

Dilute 25–30-µL mouse serum sample
to 200 µL with Buffer A. Consult cartridge certificate for true sample
capacity. Filter through 0.22-µm spinfilter.

Remove cartridge cap and plug, attach
Luer-Lock adapter to cartridge, draw
4 mL of Buffer A into syringe and dispense through cartridge via Luer-Lock,remove excess Buffer A from top ofresin bed with transfer pipette.

Remove Luer-Lock adapter and add200-µL diluted serum sample. Cap cartridge loosely or leave open. Place in
1.5-mL collection tube labeled “Flowthrough fraction 1” (F1). Centrifuge
1.5 min at 100 × g.

Add 400-µL Buffer A. Centrifuge
2.5 min at 100 × g. Collect in F1 tube.

6 Prepare for elution

7 Elute bound fraction

8 Re-equilibration

9 Analyze F1 + F2

F2
Place spin cartridge in new collection
tube labeled “Flow-through fraction 2”(F2). Add 400-µL Buffer A. Centrifuge
2.5 min at 100 × g. Collect in F2 tube.

F1 + F2

Remove spin cartridge from F2 tube
and attach Luer-Lock adapter tightly to
top of cartridge.

Fill 5-mL Luer-Lock plastic syringe with
2-mL Buffer B and attach to Luer-Lockadapter. Slowly push Buffer B throughcartridge to elute bound proteins into
new collection tube. Save eluant withtargeted high-abundant proteins foranalysis or discard.

Fill new 5-mL plastic syringe with
4-mL Buffer A and attach to Luer-Lockadapter. Slowly push Buffer A throughcartridge to re-equilibrate the cartridge
for the next sample or store wettedwith Buffer A (at 4 °C). Recap bothends for storage.

Fractions F1 and F2 can be analyzed
individually or combined. Concentrate
and analyze these fractions containing
low-abundant proteins.

For more detailed
instructions or informationon accessories, refer to theAgilent Multiple Affinity
Removal Spin Cartridge
Instruction Guide

For Mouse Serum

Visit www.agilent.com/chem/bioreagents formore products that will help make yourproteomics research more efficient:- Multiple Affinity Removal LC columns forautomated serum/plasma processing- Lys Tag 4H reagent for improving MALDI-MS
analysis of peptides and proteins

Multiple Affinity
Removal LC column- Tryptic digestion kit for digesting gel-isolated
proteins
Lys Tag 4H
Reagent Kit- C18 Cleanup Pipette Tips and Spin Tubes for
purifying proteomics samples- Spin filters and concentrators for cleaning,concentrating, and buffer-exchanging
samplesProtein In-Gel
Tryptic Digestion Kit- MALDI-MS matrices and peptide/protein
standards for your proteomics studies
Cleanup C18
Pipette Tips

Agilent Multiple Affinity Removal Spin Cartridge
Part Number 5188-5289

For More Information

Quick Reference GuideFor more information on our products and services, visit our Web site atwww.agilent.com/chem/bioreagents.
Or call your local authorized Agilent distributor.

Agilent shall not be liable for errors contained herein orfor incidental or consequential damages in connection
with the furnishing, performance, or use of this material.

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Information, descriptions, and specifications in thispublication are subject to change without notice.
© Agilent Technologies, Inc. 2004
Printed in the USA
November 1, 2004
5188-5291EN

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