ProteomeLab™ Protein Method Development Guide

P/N 726468CBJanuary 2012

Beckman Coulter, Inc., 250 S. Kraemer Blvd., Brea, CA 92821

© Copyright 2012 Beckman Coulter, Inc.,

®

Copyright© Beckman Coulter, Inc., 2012. All rights reserved. No part of thispublication may be reproduced, transcribed, transmitted, or translated intoany language in any form by any means without the written permission ofBeckman Coulter, Inc.

Licenses and TrademarksBeckman Coulter® is a registered trademark of Beckman Coulter, Inc.32 Karat™ Software is a trademark of Beckman Coulter, Inc.

Microsoft® is a registered trademark and Windows™ and Windows NT™are trademarks of Microsoft Corporation.

TEFZEL®, Teflon®, Hastelloy C® and Kel-F® are registered trademarks oftheir respective companies.ProteomeLab™ is a trademark of Beckman Coulter, Inc.

Table of Contents

Overview ........................................................................................ 1Safety Information .................................................................. 1

General Precautions ......................................................... 1 Materials & Reagents ............................................................. 2

Storage of Kit Components .............................................. 3Short Term Storage (< 24 hours) of Capillary ................. 3Long Term Storage (> 24 hours) of Capillary ................. 3

Principle of the Method .................................................................. 4Preparing the Working Solution ............................................. 5Installing the Capillary ............................................................ 7

Capillary Equilibration .................................................... 7Capillary Reconditioning ................................................. 8

Performing a Run .................................................................... 8Checking Your Results ......................................................... 11Running a Sample ................................................................. 11Troubleshooting Procedures ................................................. 15

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The ProteomeLab™ Protein Method Development Guide

1. OverviewWith capillary zone electrophoresis (CZE) proteins are separated by their electrophoretic mobility, a function of the charge and mass of a protein at a given pH. When using electrolytes with pH > 4.0, the surface of bare-fused silica becomes negatively charged, inducing both electroosmotic flow (EOF) and the adsorption of proteins that possess a net positive charge. The Protein Methods Development kit, contains coated capillaries, buffers, standards and EOF markers to allow you to optimize a separation method for the analysis of a broad spectrum of proteins. The use of this Neutral capillary surface serves to significantly reduce EOF and to prevent adsorption of proteins to the capillary surface. These Neutral capillaries have a two layer coating, with the first layer a bonded phase serving to deactivate the silanol groups and a second hydrophilic layer that protects against hydrophobic interactions improving the overall efficiency and resolution of proteins separated by CZE. This chemistry is designed to be used with the ProteomeLab™ PA 800 UV detector only - the higher energy generated by the PDA detector may serve to degrade the capillary surface.

Safety InformationRefer to the MSDS information included with this kit for information on the proper handling of materials and reagents and always follow standard laboratory safety guidelines.

General Precautions

• Review this manual carefully and verify that all kitcomponents are accounted for prior to beginning theprocedure.

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• Equilibrate all samples and reagents to room temperature(20°C - 25°C) before use.

• Avoid exposing samples and reagents to excessive heat orlight during storage.

• Follow the guidelines listed on the labels for properstorage requirements.

• Do not freeze capillary and buffer solutions.• Do not use the kit beyond the expiration date.

Materials & Reagents

Materials Provided in Kit (Reorder# 477445)

NOTE: Performance specifications of this product is based onusing the buffers supplied in this kit or replacementssupplied by Beckman Coulter only.

Materials Needed but Not Provided in Kit

Component Quantity Reorder #Neutral Capillary, 50 µm 1 P/N477441

Orange G Reference Marker, 0.1% aqueous solution 1 mL P/N241524

Histamine Reference Marker, 1% aqueous solution 1 mL P/N477446

Citrate Buffer, pH 3, 50 mM 100 mils P/N477442

Citrate/MES Buffer, pH 6, 50 mM 100 mils P/N477443

Tricine Buffer, pH 8, 50 mM 100 mils P/N477444

Protein Test Mix (contains 1 mg each of Lysozyme, Ribonuclease A, and 3 mg Cytochrome C)

1 P/N477436

DescriptionBEC Part Number

ProteomeLabTM PA 800

2mL glass vials (pack of 100) P/N144980

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P/N - Part Number

Storage of Kit Components

• Upon receipt, store all components at 2°C-8°C.• Make aliquots of the reconstituted test mix and store at -

20°C. Avoid repeated freezing and thawing.• Once the capillary has been used, it should be stored as

described below.

Short Term Storage (< 24 hours) of Capillary

1. Perform a 5-minute rinse (30 psi) with water.2. The capillary may be stored on the instrument with

capillary ends immersed in water.3. Perform a 5-minute rinse (30 psi) with water before

performing a separation, whenever the capillary has notbeen used for 3 hours or longer.

Long Term Storage (> 24 hours) of Capillary

1. Perform a 1-minute (30 psi) rinse with 0.1 N HCl. Followthis with a 3-minute (30 psi) rinse of fresh Citrate/MESBuffer, pH 6.0.

Vial caps for 2mL glass vials (pack of 100) P/N144648

Micro vial springs – required to use the PCR vials (pack of 10) P/N358821

PCR vial holder (pack of 50) P/N144657

PCR vials (pack of 50) P/N144709

PCR vial caps – gray caps (pack of 50) P/N144656

Blank capillary cartridge

CE grade water (filtered through 0.2 µm pore filter)

0.1 N HCl

0.2 µm nylon syringe filter

1000 µL pipette

100 µL pipette

10 µL pipette

DescriptionBEC Part Number

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2. Remove the capillary from the instrument and place incassette box with capillary ends placed in vials of freshCitrate/MES Buffer, pH 6.0. Store cassette box at 2°C to8°C in an upright position.

3. Perform a 5-minute rinse (30 psi) with water beforeperforming a separation, whenever the capillary has notbeen used in a while.

2. Principle of the MethodThe information presented here is intended as a general guide to help the user develop methods to separate proteins using a coated capillary designed to reduce protein adsorption and minimize electroosmotic flow. While procedures suggested may be expected to give good results in many applications, variables such as temperature, concentration, time, capillary length, voltage and pH may need to be altered to optimize a separation for a particular application.

The Neutral Capillary is designed to deactivate the silanol groups on the silica capillary wall, thereby reducing the electrostatic interactions between proteins and the capillary wall. Thus, the electroosmotic flow (EOF) of the coated capillary is extremely slow (less than 5% of the EOF determined for bare fused silica). The pH 3 and 6 buffers provided in this kit are used for the separation of proteins with isoelectric points greater than 4.0 and 6.7, respectively, using normal polarity (inlet is on the cathode side). The pH 8 buffer is used for the separation of proteins with isoelectric points less than 6.7, using reverse polarity. It is, therefore, necessary to determine the pH of the run buffer and set the polarity before starting a run (see Table 1).

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Table 1. The applicable pH range and corresponding marker for each buffer provided in the kit.

NOTE: The capillary is designed to be effective between pH 3and 8. Use of buffers outside this pH range mayshorten the life of the capillary.

Capillary Zone Electrophoresis generally uses an electric field in the range of 250 to 500 V/cm. Since the EOF of the Neutral Capillary is negligible, it is recommended that a high voltage be used in order to obtain the greatest peak efficiencies.

NOTE: The maximum electric field the coated capillary cantolerate is 500 V/cm. The use of field strength higherthan this value will result in shorter lifetime of thecapillary.

Although the Neutral Capillary is coated with a hydrophilic layer that reduces the adsorption of proteins on the capillary wall, sample clean-up may still be necessary prior to injection. Also, if peak broadening begins to occur, or if migration times begin to increase, the capillary surface may be regenerated by rinsing the capillary with 0.1 N hydrochloric acid for 30 to 60 seconds prior to performing a 1.5-minute rinse with buffer.

Preparing the Working Solution

Preparing the Test Mix

NOTE: The Test Mix is intended to be used to check theperformance of the capillary at pH 3 and 6 usingnormal polarity. No test mix is supplied to test theseparation using reverse polarity at pH 8, but injection

Buffer pHReference Marker

PolarityApplicable pH

Range

3 Histamine Normal > 4.0

6 Histamine Normal > 6.7

8 Orange G Reversed < 6.7

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of 0.01% Orange G Reference Marker should give asharp peak at 3-4 minutes under these separationconditions.

1. Add 1 mL of water to the dried Test Mix and mix welluntil dissolved. Set aside 100 µL in a 200 µL PCR vial touse in step 2 below. Make 100 µL aliquots of theremaining solution and store promptly at - 20°C.

2. Add 10 µL Histamine Reference Marker to the Test Mixsample and mix well.

3. Put the vial into a PCR vial holder with spring and graycap (see Figure 1).

Figure 1. The assembly of a PCR vial and spring within a PCR vial holder.

Preparing the Sample

1. The suggested protein sample concentration is between 50and 1000 µg/mL for optimum separations. This suggested

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range may vary depending upon the selected wavelengthand buffers used.

2. Add 10 µL of Reference Marker to 100 µL of samplesolution. Use the Histamine Reference Marker if theCitrate Buffer (pH 3) or the Citrate/MES Buffer (pH 6)will be used for the separation. Use the Orange GReference Marker if the Tricine Buffer (pH 8) will be run.

Preparing the Buffers Vials

Fill three 2mL glass sample vials with the selected buffer. Use the Citrate/MES Buffer (pH 6) to separate the Test Mix. Sonicate or degas the buffer to remove small bubbles that might interfere with the separation.

NOTE: Buffer solutions may promote microbiologicalgrowth. If signs of cloudiness appear in the buffer,filter the buffer through 0.2µm filter prior to use.

Installing the CapillaryBefore a coated capillary is used on the instrument, it must be installed in a cartridge according to the instructions contained in the Capillary Replacing Procedure. The length of the capillary used will depend on the separation requirements and nature of the samples. In general, samples are well separated on a 30.2cm capillary. The use of 40.2cm capillary may have better resolution at an expense of longer separation time.

NOTE: After the cartridge is installed, it is critical to theperformance of the coated capillary that the ends notbe exposed to air for longer than 20 minutes to avoiddrying. Be sure to immerse the ends of the capillary invials of water.

Capillary Equilibration

Prior to using the capillary for the first time, perform a rinse for 1 minute (30 psi) with 0.1N HCl. Follow this with a 10-minute rinse (30 psi) with the selected pH buffer. Equilibrate the

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capillary with the buffer by applying 500 V/cm for 10 minutes. Follow this equilibration with a 10 minute buffer rinse (30 psi).

Capillary Reconditioning

When changing to a buffer of a different pH, recondition the capillary by performing a 10-minute rinse (30 psi) with the new buffer. Allow the new buffer to set in the capillary, without voltage, for 10 minutes prior to performing a run.

Performing a Run 1. Clean the electrodes, injection ports and capillary ends to

ensure good reproducibility of migration time.2. Program a test method using the following example

separation conditions.

Panel A

Capillary Temperature 20oC

Sample Storage Temperature 10oC

Collect Current Trace

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Panel B

UV Detection

Wavelength 214 nm

Data Rate 2 hz

Filter Normal

Peak Width more than 25

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Panel C

Figure 2.0 Programming the System Test RunPanel A: System Initial ConditionsPanel B: UV Detector Initial ConditionsPanel C: Time Programming3. Prepare Test Mix, Citrate/MES Buffer (pH 6) vials, as

well as 0.1 N HCl rinse buffer and water.4. Place the vials in the following positions:

Acidic Rinse 0.1 N HC1 wash, 0.5 min. at 30 psi

Water Rinse Water wash, 1.5 min. at 30 psi

Sample Injection 0.5 psi for 3 seconds

Voltage Separation 15 kV for 25 min., 0.17 min. ramping time

Water Rinse Water wash, 1 min. at 30 psi

Buffer vials (3 vials) BI:A2, BI:C2, BO:A2Waste BO:B10.1N HC1 BI:B2Water (2 vials) BI:A1, BO:A1Test Mix vial SI:A1

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5. Prepare and rinse the capillary with CE grade water asdescribed under “Installing the Capillary.”

6. Run the test method.

Checking Your ResultsA typical result of the test run is shown in Figure 3 with the corresponding current shown in Figure 4. The first peak, Histamine Reference Marker, normally migrates at 3-4 min. For best migration time reproducibility, the run buffer vials should be replaced every 20 runs and the rinse buffer vial refilled as needed. The reproductivity of an analytes mobility for separations made from this kit is typically less than 2% relative standard deviation, run-to-run, day-to-day.

Running a SampleSamples are run as described in the section “Performing a Test Run” with the following alterations:

1. Set the polarity in the Time Program as required. If theTricine Buffer, pH 8, is used to separate a negativelycharged sample, the polarity must be reversed (inlet on the

Buf HCL Buf

H2O Waste

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5

4

3

2

1

(Left) Inlet Buffer TrayBI

B C D E FA B C D E FA

Outlet Buffer Tray (Right)BO

Buf

H2O

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cathode side) so that the analyte will migrate past thedetector.

2. Place the 2mL vials filled with the selected buffer inposition.

3. Place the PCR vial with the sample into a PCR vial holderwith spring (see Figure 1).

4. Adjust the inject time or pressure in the Time Programwith the separation method to give sufficient signalintensities of peaks.

5. Recondition the capillary with the selected buffer asdescribed under “Capillary Reconditioning.”

6. Perform the run.

NOTE: Some proteins might interact with histamine ororange G, leading to broad and/or split peaks with themarkers. When observing the latter phenomenon,separately inject the sample and reference markersinto the capillary, instead of injecting the mixture ofsample and reference marker.

Figure 3. Electropherogram of the test run.

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Figure 4. Current of the test run.

NOTE: When using the Citrate Buffer, pH 3.0, with a 30.2 cmcapillary and an electric field of 500 V/cm, themigration time of the Histamine Reference Markershould be 2-3 minutes.

An example of separation of immunoglobulin (IgG) is shown in Figure 5 with the Citrate Buffer, pH 3.0, a 30.2 cm capillary, and an electric field of 500 V/cm.

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Figure 5. Electropherogram of capillary electrophoretic separation of IgG sample with the use of Citrate Buffer, pH 3.0.

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Troubleshooting Procedures

Problem Possible Cause Corrective Action

Unsteady or low current (< 5 µA for Citrate/MES Buffer or Citrate Buffer and < 3 µA for Tricine Buffer on 30.2 cm capillary and 500 V/cm).

Capillary is plugged. Terminate the run, and perform 5-minute pressure rinse (40 psi) with water to flush the capillary from both ends.

Broken capillary. Replace the capillary.

Air in capillary. Be sure that buffer vials are adequately filled.

Contamination on the electrode.

Clean the electrodes and injection port.

High current or spikes in current (> 8 µA for Citrate/MES or Citrate Buffer and > 6 µA for Tricine Buffer on 30.2 cm capillary and 500 V/cm).

Contaminated buffer. Replace buffer as needed.

Contamination on electrode.

Clean electrodes and injection port.

HCl still in capillary during the run

HCl still in capillary during the run

Inadequate conditioning of capillary.

Repeat conditioning with selected buffer.

Poor migration time reproducibility.

Material adsorbed onto capillary coating.

Rinse the capillary with 0.1 N HCl for 30 to 60 seconds prior to performing a 1.5-minute rinse with buffer

Aged buffer solution or samples.

Replace buffer or sample vials.

Plugged capillary. Terminate the run, and perform 5-minute pressure rinse (40 psi) with water to flush the capillary from both ends.

Contamination on electrodes and/or build-up of material on the injection port.

Clean electrodes and injection port.

Deteriorated capillary. Replace the capillary.

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Broad peaks, tailing or low efficiency.

Deteriorating sample or buffer.

Replace buffer or sample as needed

Material adsorbed onto capillary coating.

Rinse the capillary with 0.1 N HCl for 30 to 60 seconds prior to performing a 1.5-minute rinse with buffer

Contamination on electrode.

Clean electrodes and injection port.

Deteriorated capillary. Replace the capillary.

No peak or low signal intensities.

Polarity of instrument. Use appropriate polarity

Capillary is plugged. Terminate the run, and perform 5-minute pressure rinse (40 psi) with water to flush the capillary from both ends.

Spikes in electropherogram.

Air in the buffer. Make sure buffer is at room temperature and air is removed by degassing or sonicating.

Problem Possible Cause Corrective Action

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