ProteomeLabTM SDS-MW Analysis Guide 390963AC SD… · SDS-MW Separation Method ... linear with its reciprocal electrophoretic mobility. ... Internal Standard 10 kDa protein 5mg/mL

ProteomeLabTM

SDS-MW Analysis GuideP/N 390963AC

December 2009

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

© Copyright 2004 Beckman Coulter, Inc.

®

Copyright

© Beckman Coulter, Inc., 2009. All rights reserved. No part of thispublication may be reproduced, transcribed, transmitted, ortranslated into any language in any form by any means without thewritten permission of Beckman Coulter, Inc.

Licenses and Trademarks

Beckman Coulter® is a registered trademark of Beckman Coulter,Inc.32 Karat™ Software is a trademark of Beckman Coulter, Inc.ProteomeLab™ is a trademark of Beckman Coulter, Inc.

Table of Contents

Overview ........................................................................................ 1Protein Size Standard .............................................................. 2Internal Standard ..................................................................... 2

Materials & Reagents .................................................................... 2Kit and Component Storage ........................................................... 3

Reagents .................................................................................. 3Capillary .................................................................................. 3

Prepare SDS-MW Size Standard and Protein Sample ................... 4 Preparation of SDS-MW Size Standard ................................. 4Preparation of Protein Sample ................................................ 5

Desalting the Protein Sample ........................................... 5Protein Sample Concentration ......................................... 5Reducing the Protein Sample ........................................... 6Non-reduced Protein Sample Preparation ........................ 6

Install Capillary Cartridge .............................................................. 7Prepare CE Instrument ................................................................... 7Perform CE Run ............................................................................. 8

Tray configuration ........................................................... 9Prepare sample for each injection. ................................. 10Capillary Pre-conditioning Method ............................... 11SDS-MW Separation Method ........................................ 14Check Separation Results .............................................. 17Estimation of Protein Molecular Weights ..................... 18

Troubleshooting Guide ................................................................. 19

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SDS-MW Analysis Kit

1. OverviewCapillary electrophoresis (CE) has become an effectivereplacement for manual slab gel electrophoresis processes dueto its automation, quantitation, fast speed and high efficiency.Many biomolecules, such as proteins, carbohydrates andnucleic acids are separated by molecular sievingelectrophoresis using gel matrices, a technique referred to ascapillary gel electrophoresis (CGE). The separation resultsfrom analytes’ differential migration through a gel matrix. Inthis case, smaller molecules will move faster than largemolecules through the separation gel. For polypeptides andproteins, it is necessary to denature the sample in the presenceof SDS, an anionic detergent that binds the proteins in aconstant ratio of 1:1.4 of protein. The constant mass-to-chargeproperty of the SDS-bound proteins allows separationaccording to differences in protein molecular weight.

The ProteomeLab™ SDS-MW Analysis Kit is designed for theseparation of protein-SDS complexes using a replaceable gelmatrix. The gel is formulated to provide an effective sievingrange of approximately 10 kD to 225 kD (Figure 1). Withinthis size range, the logarithm of protein molecular mass islinear with its reciprocal electrophoretic mobility. So themolecular weight of an unknown protein may be estimatedfrom a standard curve of known protein sizes. This kit can alsobe used to quantify the amount of protein and to determine thepurity of a protein product.

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Protein Size StandardThe SDS-MW Size Standard contains 10, 20, 35, 50, 100, 150and 225 kDa proteins. The SDS-MW size standard is used tocalibrate the gel to estimate the protein molecular weight ofyour sample. It also provides confirmation of the resolvingpower of your experiment.

Internal StandardA 10 kD protein is used as a mobility marker. The mobility ofall protein samples are calculated relative to this mobilitymarker allowing for more accurate size estimation and analyteidentification.

2. Materials & Reagents

Contents of this Kit (reorder # 390953)

Component Quantity Reorder #

Capillary 57 cm, 50 µm I.D. bare-fused silica

2 390953

SDS-MW Gel Buffer - proprietary formulation

140 mL 390953

SDS Sample Buffer - 100 mM Tris-HCl pH 9.0/1%SDS

50 mL 390953

SDS Protein Sizing Standard (10 to 225 kDa) 16 mg/mL

100µL 390953

Internal Standard 10 kDa protein 5mg/mL 0.4 mL 390953

Acidic Wash Solution 0.1 N HCl 100 mL 390953

Basic Wash Solution 0.1 N NaOH 100 mL 390953

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Materials Required but Not Provided in the Kit

3. Kit and Component Storage

ReagentsUpon receipt, store protein Sizing Standard and internalstandard at 2°C to 8°C. The Capillary, Sample Buffer, and GelBuffer may be stored at room temperature. If precipitation isnoted in SDS Gel Buffer and sample buffer, stir untilprecipitation is completely dissolved.

CapillaryOnce the capillary has been used for separation, it should bestored as follows:

ComponentBeckman

Coulter Part Number

0.5mL micro-centrifuge capped vials (pack of 500) 344319

CE grade water, DI water filter through a 0.22 µL filter

2-mercaptoethanol

Water bath (37°C to 100°C)

Microcon YM-10 Centrifugal Filter Unit

Centricon YM-10 Centrifugal Filter Unit

2mL glass vials (pack of 100) 970657

Vial Caps for the 2mL glass vials – red caps (pack of 100)

144648

200µL PCR vials (pack of 50) 144709

Micro vial springs - required to use the PCR vials (pack of 10)

358821

PCR Vial Holders (pack of 50) 144657

PCR Vial Caps – grey caps (pack of 50) 144656

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1. For long term storage, we recommend the use of avigorous cleaning method (See deep cleaning method intable below) followed by filling the capillary with freshSDS-gel. Remove capillary from instrument. Store thecapillary in the cartridge box with CE grade water vials onthe inlet and outlet ends of the capillary. Keep capillarycartridge in the cartridge box in an upright position.

2. For short term storage on instrument (< 5 days), alsoperform a deep cleaning method and fill capillary withSDS gel. The capillary end should rest in SDS gel buffer.

4. Prepare SDS-MW Size Standard and Protein Sample

Preparation of SDS-MW Size Standard 1. Remove SDS-MW Size Standard from refrigerator. Leave

at room temperature for 15 min. before starting samplepreparation.

2. Mix the standard thoroughly and centrifuge in a standardmicrofuge briefly.

3. Pipette 10 µL of Size Standard into a micro vial.4. Add 85 µL of Sample Buffer into the micro vial.5. Add 2 µL of Internal Standard into micro vial.6. Add 5 µL 2-mercaptoethanol. Cap the vial tightly and then

mix thoroughly.

Deep cleaning method

Basic Rinse 0.1 N NaOH basic wash, 10 minute at 50 psi

Acidic Rinse 0.1 N HCl acidic wash, 5 minutes at 50 psi

Water Rinse CE grade H2O, 2 minutes at 50 psi

Gel Rinse SDS gel buffer, 10 minutes at 50 psi

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7. Heat mixture in a water bath at 100°C for 3 minutes in aclosed microvial.

8. Place the vial in a room temperature water bath to cool for5 minutes before injection. The sample will remain stablefor approximately 24 hours.

Preparation of Protein Sample Desalting the Protein Sample

The signal intensity and resolution of this kit are sensitiveto the salt concentration in the protein sample. Generally,if the final salt concentration is above 50 mM, the sampleloading efficiency will be reduced. The sample should bedesalted with a Centricon column using the followingprocedure:

1. Add 1 mL of protein sample to Centricon YM-10, thenadd 1 mL SDS sample buffer into Centricon.

2. Centrifuge at 7,000 g for 20 min.3. Add 2 mL of sample buffer, then centrifuge at 7,000 g for

20 min.4. Insert the centricon upside-down to drain the suspended

protein solution (in the filter membrane) into a new vialand centrifuge for 3 min. at 5,000 g.

5. Transfer the collected protein to an appropriate steriletube. Add sample buffer to give a final volume of 1mL.

Protein Sample Concentration

After the addition of the SDS sample buffer, the total proteinconcentration should be within the range of 0.2 mg/mL to 2mg/mL. For best results, the recommended proteinconcentration is 1 mg/mL. If the protein concentration is toohigh, it may result in insufficient SDS binding, giving broadpeaks and poor resolution. On the other hand, if the proteinconcentration is too low, a low signal will be observed.

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Reducing the Protein Sample

Reduction of the disulfide bonds will provide a more accurateassessment of a protein’s molecular weight and will allow youto gain additional structural information on a given protein. Inthis case:.

1. Dilute the sample with the SDS sample buffer for a total95 µL volume to give a final protein concentration range of0.2 mg/mL to 2 mg/mL.

2. Add 2 µL of Internal Standard.3. Add 5 µL of 2-mercaptoethanol. Cap the vial tightly and

then mix thoroughly. 4. Heat mixture in a water bath at 100°C for 3 minutes in a

closed micro vial. 5. Place the vial in a room temperature water bath to cool for

5 minutes before injection.

Non-reduced Protein Sample Preparation

Comparison of a protein’s reduced versus non-reduced statecan yield important structural information. In this case it is agood idea to alkylate your protein sample to minimize anyheterogeneity created from partial auto-reduction of yourprotein. The following procedure is recommended

Preparing Alkylation Reagent

A 250 mM solution of Iodoacetamide (IAM) is recommendedas an alkylation reagent. The solution is stable forapproximately 24 hours at room temperature.

1. Weigh out 46 mg IAM and add into microvial. 2. Add 1 mL of CE grade H2O into sample vial, cap the vial

tightly and then mix thoroughly.

Preparing the Sample

1. Dilute the sample with the SDS sample buffer for a total95 µL volume to give a final protein concentration range of0.2 mg/mL to 2 mg/mL.

2. Add 2 µL of Internal Standard.

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3. Add 5 µL alkylating reagent. Cap the vial tightly and thenmix thoroughly.

4. Heat mixture in a water bath at 70°C for 3 minutes in aclosed micro vial.

5. Place the vial in a room temperature water bath to cool for5 minutes before injection.

5. Install Capillary CartridgeInstall a fused silica capillary into a cartridge according to thecapillary replacement procedure in the system manual. TheSDS-MW assay was optimized using a 30.2 cm capillary with20.2 cm from the inlet to the detection window. The migrationtime of protein molecules depends on the capillary length for agiven voltage. In order to get good reproducibility fromcapillary to capillary, it is recommended that the capillaryshould be pre-measured to exact length and then installed inthe cartridge.

NOTE: The cut ends of capillaries should be inspectedcarefully under magnification. The cut must beclean (not jagged) and perpendicular to thecapillary length (not angled). Poor cuts will resultin poor resolution and poor sample loading.

6. Prepare CE InstrumentPerform system tests as described in your manual to determinethe suitability of your system prior to analysis.

NOTE: Carefully clean capillary tips, electrodes andinterface block with a Kim-wipeTM soaked withCE grade water after every 15 hours of operation,or when shutting down or starting up for the newrun sequence. The SDS gel buffer is very viscousand will accumulate on the tips, electrodes andinterface block if regular thorough cleaning is notemployed.

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7. Perform CE Run This method introduces the sample from the left tray of thesystem, generating an inlet to detection window distance of20.2 cm. A sequence template is set up for automatic runsequence of 24 samples. The CE analysis is based on six-runcycles. Each six-run cycle uses SDS gel buffer on bothcapillary inlet and outlet. Fill the buffer vials with the SDS GelBuffer, 0.1 N NaOH basic wash solution, 0.1 N HCl acidicwash solution, and water according to the instructions below.After filling the vials with SDS gel, degas the vials for 5minutes under 5 to 15 Hg vacuum. The waste vial should beempty. To avoid carryover on the capillary outlet, replace thewaste vial every time when starting a new sequence.

NOTE: When filling the vial with SDS gel, be careful notto get the gel on the inside vial rim. It isnecessary that the inside vial rim be clean and dryto allow the vial cap to seat properly.

Figure 1. Filling 2.0 mL Vials

Gel Fill Level

2.0 mL Vial

Red Cap

900037L.AI

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All solutions should by replaced after six cycles. This may bedone automatically using the increment function of the systemsoftware. The inlet and outlet tray configurations, shown inFigures 2.0 and 3.0, are set up to run 24 cycles. If fewer cyclesare planned, adjust the number of reagents accordingly. Eachsample or sample repetition will constitute a cycle. An exampleof a sequence set-up for 24 cycles is shown in Figure 7.0

Tray configuration

Inlet buffer tray

Figure 2.0 Left Tray (inlet) configuration

A1 to A5: CE grade H2O, use for dip step to clean capillary tip

C1 to C4: SDS gel for separation

D1 to D4: 0.1N NaOH, use to precondition capillary

E1 to E4: 0.1N HCl, use to precondition capillary

F1 to F4: CE grade H2O, use for capillary preconditioning

H2O

H2O

H2O

H2O

H2O Gel

Gel

Gel

Gel NaOH

NaOH

NaOH

NaOH

HCl

HCl

HCl

HCl

A B C D E F

H2O

H2O

H2O

H2O

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Outlet buffer tray

Figure 3.0 Right tray (outlet) configuration

Prepare sample for each injection.

Aliquot 100 µL sample into micro sample vials. Set the samplevials into the inlet sample tray from A1 to C8 following theinstructions shown in Figure 4.0. For the best quantitation, it isrecommended to perform only one injection per vial. Figure4.0 illustrates the correct assembly of the microvial holders.

A1 to A5: CE grade H2O, use in dip step to clean capillary tip

B1 to B4: Empty vial with 1.0 ml of CE grade water for SDS gel rinse

C1 to C4: SDS gel for separation

D1 to D4: Empty vial for 0.1N NaOH rinse

E1 to E4: Empty vial for 0.1N HCl rinse

F1 to F4: Empty vial for CE grade H2O rinse

H2O

H2O

H2O

H2O

H2O Gel

Gel

Gel

Gel

A B C D E F

Waste

Waste

Waste

Waste

Waste Waste Waste Waste

Waste Waste Waste

Waste Waste

Waste

Waste

Waste

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Figure 4.0 Installation of Sample Vial

Capillary Pre-conditioning Method

The methodology is based on six-run cycles. Prior to the startof each six-run cycle a capillary pre-conditioning methodshould be run. This method serves to clean the capillary andequilibrate the capillary and its surface with a fresh polymersolution. For the best results we recommend running the pre-conditioning step twice before each cycle of six sample runs.Preparing the pre-conditioning method is highlighted in Figure5.0

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Panel A

Panel B

* Basic Rinse 0.1 N NaOH basic wash, 5 minute at 50 psi

* Acidic Rinse 0.1 N HCl acidic wash, 2 minutes at 50 psi

* Water Rinse CE grade H2O, 2 minutes at 50 psi

* SDS Gel Rinse SDS gel fill, 10 minutes at 40 psi

* Pre-run 15 kv for 10 min., 5 min. ramping time

* Repeat this step every six cycles

Capillary Temperature 25oC

Sample storage Temperature 25oC

Constant Voltage collect current trace

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Panel C

Figure 5.0 32 Karat Software, Version 7.0, Pre-conditioningMethod

Panel A - Highlights the time-programming set-up

Panel B - Highlights the system initial conditions

Panel C - Highlights the PDA detector initial condition

Detection Photo Diode Array

Wavelength 220 nm

Bandpass 10 nm

Reference Channel 300 nm 10nm bandwidth

Data Rate 2 hz

Detection Filter Normal

Peak width (points) 16-25

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SDS-MW Separation Method

Panel A

Panel B

Water Dip water dip for 0.0 minute

Water Dip water dip for 0.0 minute

Sample Injection 20 second at 5 KV,

Voltage Separation 30 minutes at 15 kV (reverse polarity), 1 minute ramp, Applying 20 psi to both inlet and outlet

Capillary Temperature 25oC

Sample Storage Temperature 25oC

Constant Voltage Collect Current

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Panel C

Panel D

Detection Photo Diode Array

Wavelength 220 nm

Bandpass 10 nm

Reference 300 nm 10 nm bp

Data Rate 2 hz

Detection Filter Normal

Peak width (points) 16-25

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Panel E

Figure 6.0 32 Karat Software, Version 7.0, SDS-MWSeparation Method

Panel A: Highlights the Time programming set-up

Panel B: Highlights the system initial conditions

Panel C: Highlights the PDA detection initial conditions

Panel D: Highlights the recommended integration events

Panel E: Highlights the recommended peak identification parameters for the protein molecular weight ladder

Sequence Table

Figure 7.0 Example of Sequence Set-up for 24 sample cycleset

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Check Separation Results

The Protein Sizing Standard Test Mix contains 7 proteins(10kD, 20kD, 35kD, 50kD, 100kD, 150kD and 225kD). Allproteins should be completely separated within 30 minutesusing our recommended method. A typical separation of theSizing Standard Test Mix is shown here as an example, Figure8.0.

Figure 8.0 Separation of Protein Molecular Weight SizeStandard

8 10 12 14 16 18 20 22 24 26 28 30

Minutes

0.00

0.005

0.010

0.015

0.020

0.025

0.030

0.035

AU 10 kD

20 kD

35 kD

50 kD

100 kD

150 kD

225 kD

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Estimation of Protein Molecular Weights

A 10kD mobility marker is used as an internal referencestandard and all the protein mobility is calculated relative tothis internal standard. Figure 9.0 is a typical calibration curveobtained by plotting the Log of molecular weight vs. mobilityof each protein in the protein Sizing Standard (1/X). Themolecular weight of an unknown protein can be estimated byusing this calibration curve. A quadratic curve-fittingrelationship will typically give you the best curve-fit. Werecommend that you re-calibrate this curve every 24 cycles.This is accomplished by running the MW sizing standard andupdating the mobility values for each standard to reflect thenew run. This update is performed in the qualitative analysis ofyour 32 Karat Version 7.0 Control and Analysis Software.Figure 10.0 shows an example of this set-up.

Figure 9.0 Calibration Curve of Protein Molecular Weight SizeStandard

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Figure 10.0 Quantitative Analysis Screen. An example of theset-up for the qualitative analysis tab to update the sizecalibration curve.

8. Troubleshooting Guide

Problem Possible Cause Corrective Action

Low or unsteady current Capillary Plugged (1) Rinse the capillary with CE grade water at 100 psi for 10 min. and then perform capillary deep cleaning method. (2) If unsteady current still occurs after step (1), change to a new capillary.

Air bubble in the gel 1) Degas SDS gel under 5 to 15 Hg vacuum for 5 minutes.

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High current Contaminated Gel Buffer Replace gel buffer as needed

Contamination on electrode

Clean electrodes, (see Instrument Manual).

Spikes in electropherogram

Air bubble in gel buffer 1) Degas SDS gel under 5 to 15 Hg vacuum for 5 minutes.

Broad peaks or low efficiency

Poor capillary end cut Check the capillary end under 10x magnification. Re-cut the capillary end if the cut is not clean or is angled.

Improper reduction of sample

Reduce sample using recommended procedure. Use fresh mercapitoethanol for sample reduction.

High salt in protein sample Desalt sample using recommended procedure.

Deteriorated capillary Replace capillary when other attempts to reduce peak broadening fail.

No peaks Capillary inlet is longer that the inlet electrode.

Re-cut capillary inlet to make sure that it is shorter than the electrode.

Dirty or plugged capillary tip

(1) Clean capillary tip using CE grade water.(2) Re-cut the tip if the capillary was partially plugged.

Insufficient quantity of sample

Increase sample volume in the micro vial to 100 uL.

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