ProteomeLabTM SDS-MW Analysis Guide 390963AC SD… · SDS-MW Separation Method ... linear with its...
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Transcript of ProteomeLabTM SDS-MW Analysis Guide 390963AC SD… · SDS-MW Separation Method ... linear with its...
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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