QIAxcel Protein Handbook 2nd Edition April 2013

QIAGEN Sample and Assay Technologies QIAGEN is the leading provider of innovative sample and assay technologies, enabling the isolation and detection of contents of any biological sample. Our advanced, high-quality products and services ensure success from sample to result. QIAGEN sets standards in:

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Purification of DNA, RNA, and proteins

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Nucleic acid and protein assays

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microRNA research and RNAi

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Automation of sample and assay technologies

Our mission is to enable you to achieve outstanding success and breakthroughs. For more information, visit www.qiagen.com.

Contents Kit Contents

4

Storage

4

Intended Use

5

Safety Information

5

Quality Control

6

Introduction

7

Principle and procedure

7

Equipment and Reagents to Be Supplied by User

9

Important Notes

10

Access to manual and context sensitive help

10

Sample labeling

10

QX Pro Alignment Marker

10

Cartridge calibration

10

Equilibration of components

10

Reconstitution of reagents and marker

11

Protocols 

System Preparation

12

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Preparation of Samples and Marker

15

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Starting a Sample Run

17

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Analysis of Samples

19

Creating a protein size marker reference table

22

Determination of protein mass size and quantity

26

Troubleshooting Guide

28

Appendix A: Analysis of Samples >100 kDa in Size

32

Appendix B: Tips and Tricks for Protein Analysis

32

Appendix C: QIAxcel Protein Methods

38

Appendix D: Removing Gel from Blocked Capillaries

39

Standard gel-droplet test

40

Gel-droplet test with hot water

40

Appendix E: Performing a signal check

41

Ordering Information

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Kit Contents QIAxcel Protein Kit

(1200)

Catalog no.

929204

Number of runs

12 x 100

QIAxcel Protein Cartridge (with smart key)

1

QX Pro Separation Buffer

40 ml

QX Wash Buffer

40 ml

QX Mineral Oil

50 ml

QX Pro Labeling Buffer

25 ml

QX Pro Calibration Marker

0.6 ml

QX Pro Reduction Agent*

75 mg

QX Pro Alignment Marker

1.5 ml

QX Pro Marker 14–97 kDa

575 µg

QX Pro Labeling Dye

250 µg

QX 0.2 ml 12-Tube Strips

2

QX Colored 0.2 ml 12-Tube Strips (Colored)

5

Quick-Start Protocol

2

* Contains DL-dithiothreitol; see “Safety Information” on page 5.

Storage The QIAxcel Protein Kit (1200) (cat. no. 929204) comprises the QIAxcel Protein Cartridge Kit and the QIAxcel Protein Reagent Kit. The QIAxcel Protein Reagent Kit includes marker, reduction agent, and dye. These components should be stored at 2–8°C and are stable until the expiration date under these conditions. IMPORTANT: After reconstitution, store the QX Pro Reduction Agent and the QX Pro Marker 14–97 kDa at –20°C and the QX Pro Labeling Dye at 2–8°C. The reconstituted components are stable until the expiration date stated on the label. All other components delivered with the QIAxcel Protein Cartridge Kit should be stored dry at room temperature (15–25°C) and are stable under these conditions until the expiration date stated on the label.

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Cartridge storage If the QIAxcel Protein cartridge is being used on a daily basis, store the cartridge latched in the QIAxcel or QIAxcel Advanced instrument in the “Park Position”. Note: The QIAxcel or QIAxcel Advanced instrument must be left on if the cartridge is stored in the “Park Position” and the cartridge must be latched. Do not switch off the QIAxcel or QIAxcel Advanced instrument. If more than one QIAxcel Protein cartridge is being used on a daily basis, store the second cartridge in the QX Cartridge Stand in the dark or protected with the cover. Make sure that the cartridge stand reservoir is filled with QX Wash Buffer and covered with mineral oil and the purge port sealed. Alternatively, seal the purge port, return the QIAxcel Protein cartridge to the blister package, inserting the capillary tips into the soft gel, and store at room temperature in an upright position (see orientation label on blister package). Prior to first use or after being removed from the blister package, the QIAxcel Protein cartridge should be allowed to stand in the QX Cartridge Stand or latched in the instrument for at least 20 minutes. The QIAxcel Protein cartridge can be stored in this manner until the expiration date indicated on the kit label. IMPORTANT: Storing the QIAxcel Protein cartridge below 15°C can severely damage the cartridge.

Intended Use The QIAxcel Protein Kit is intended for molecular biology applications. This product is not intended for the diagnosis, prevention, or treatment of a disease. All due care and attention should be exercised in the handling of the products. We recommend all users of QIAGEN® products to adhere to the NIH guidelines that have been developed for recombinant DNA experiments, or to other applicable guidelines.

Safety Information When working with chemicals, always wear a suitable lab coat, disposable gloves, and protective goggles. For more information, please consult the appropriate safety data sheets (SDSs). These are available online in convenient and compact PDF format at www.qiagen.com/safety where you can find, view, and print the SDS for each QIAGEN kit and kit component.

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24-hour emergency information Chemical emergency or accident assistance is available 24 hours a day from: CHEMTREC USA & Canada  Tel: 1-800-424-9300 Outside USA & Canada  Tel: +1 703-527-3887 (collect calls accepted)

Quality Control In accordance with QIAGEN’s ISO-certified Quality Management System, each lot of the QIAxcel Protein Kit is tested against predetermined specifications to ensure consistent product quality.

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Introduction The QIAxcel Advanced system enables fully automated capillary gel electrophoresis and readily meets the requirements for high-throughput applications in biopharmaceutical or protein expression laboratories. Using LED-induced fluorescence, the system, together with the QIAxcel Protein Kit enables automated separation of protein samples ranging from 10 kDa to 200 kDa in size, with a detection limit of 2.5 ng/µl. The novel QIAxcel Protein cartridge is suitable for analysis of various protein samples such as purified proteins, crude lysates, and antibodies. The QIAxcel Protein Kit comprises the QIAxcel Protein Cartridge Kit and the QIAxcel Protein Reagent Kit, and is compatible with all generations of QIAxcel instruments. The QIAxcel Protein gel cartridge is reusable, allowing 100 runs of 12 samples. QIAxcel instruments are preinstalled with methods suitable for most applications and allow separation of 12 samples within 15–25 minutes. In addition, customized methods can also be developed; please contact QIAGEN Technical Services for more details. QIAxcel DNA and RNA Kits, which allow nucleic acid separation and quantification on the QIAxcel or QIAxcel Advanced instrument, are also available (see ordering information, page 43).

Principle and procedure The QIAxcel Advanced system uses capillary gel electrophoresis to enable fast separation of proteins based on size. Unlike traditional methods such as SDSPAGE, separation is performed in a capillary of a precast gel cartridge. After one-step sample preparation with a denaturing buffer and labeling of the protein samples with a dye that binds covalently to the lysine residues, each sample is automatically loaded into an individual capillary (according to voltage and time parameters) and voltage is applied to start separation. The negativelycharged proteins migrate through the capillary to the positively-charged end (Figure 1, page 8). As with SDS-PAGE, low-molecular-weight molecules migrate faster than high-molecular-weight molecules. As the molecules migrate through the capillary, they pass a detector that detects and measures the fluorescent signal. A photomultiplier detector converts the emission signal into electronic data, which are then transferred to the computer for further processing using QIAxcel ScreenGel® software. The proprietary gel matrix, in combination with fluorescence labeling, allows size-based separation and sensitive detection of protein samples. Powerful QIAxcel ScreenGel software provides data output in various formats such as gel view, electropherogram view, or superimposition of electropherograms. Several analysis parameters can be displayed in tabular format to enable absolute and relative quantification, direct comparison of bands of interest, and much more.

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Figure 1. Sample separation process using the QIAxcel Advanced system. Nucleic acid or protein molecules are separated according to size by applying an electrical current to a gelfilled capillary. A photomultiplier detector in the instrument detects the nucleic acid or protein molecules as they migrate towards the positively-charged end of the capillary. The data are converted to an electropherogram and a gel image by QIAxcel ScreenGel software.

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Equipment and Reagents to Be Supplied by User When working with chemicals, always wear a suitable lab coat, disposable gloves, and protective goggles. For more information, consult the appropriate safety data sheets (SDSs), available from the product supplier.

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Pipets and pipet tips

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Acetonitrile anhydrous (e.g., Sigma 271004-100 ml Acetonitrile anhydrous, 99.8 %) for reconstitution of QX Pro Labeling Dye

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Double-distilled water (for reconstitution of QX Pro Reduction Agent and for injection purposes in the buffer tray)

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12-tube strips (e.g., QX 0.2 ml 12-Tube Strip, cat. no. 929703) or 96-well plates

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Incubator for tube strips or 1.5 ml tubes (temperature 50°C)

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Centrifuge with rotor suitable for 0.2 ml strips, 96-well plates, or 1.5 ml tubes (e.g., Centrifuge 4-16 [cat. no. 81320] or Centrifuge 4-16K [cat. no. 81420])

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Optional: Additional QX Pro Labeling Buffer (cat. no. 929609) may be required to fill empty wells

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QIAxcel Advanced instrument (cat. no. 9001941) or QIAxcel instrument

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QIAxcel ScreenGel Software version 1.1.0 or higher

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Important Notes Access to manual and context-sensitive help Press F1 when working in QIAxcel ScreenGel software to open the software section of the instrument user manual and context-sensitive help. The user manual contains comprehensive information on QIAxcel ScreenGel software and provides detailed information on various features and options.

Sample labeling Add QX Pro Labeling Dye shortly before starting the labeling reaction. Labeled samples can be kept at room temperature (15–25°C), protected from light exposure, but must be analyzed within 8 hours of the labeling reaction.

QX Pro Alignment Marker QX Pro Alignment Marker should be replaced after every 24 runs or 30 days, whichever comes first. When not in use, tube strips containing alignment marker should be stored at –20°C.

Cartridge calibration Prior to the first sample run using a new cartridge, perform cartridge calibration. Repeat cartridge calibration when in doubt about the intensities of individual channels. Note: If the cartridge is being used on a different QIAxcel or QIAxcel Advanced instrument, or if a different computer is used to operate the instrument, the cartridge calibration has to be repeated for this particular cartridge/instrument combination. If a different computer is being used to operate the QIAxcel or QIAxcel Advanced instrument, the calibration log file must be transferred to the new computer so that calibration does not need to be performed again. See the instrument user manual for more details.

Equilibration of components Wait for complete thawing of frozen components and vortex before use.

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Reconstitution of reagents and marker QX Pro Reduction Agent

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Reconstitute QX Pro Reduction Agent with 1875 µl distilled water and store at –20°C until it is required for use.

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QX Pro Reduction Agent can be stored at –20°C until the expiration date stated on the label.

QX Pro Marker 14–97 kDa

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Reconstitute QX Pro Marker 14–97 kDa with 500 µl QX Pro Labeling Buffer and store at –20°C until it is required for use.

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Store at –20°C until the expiration date stated on the label.

Note: After reconstitution, these reagents can be stored in aliquots and rethawed when required. QX Pro Labeling Dye

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Reconstitute QX Pro Labeling Dye with 1250 µl Acetonitrile anhydrous p.a. and store at 2–8°C until the expiration date stated on the label.

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Protocol: System Preparation This protocol enables preparation and setup of the cartridge and the buffer tray, as well as calibration of the cartridge, prior to running samples. Important points before starting

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For information on the QIAxcel or QIAxcel Advanced, please refer to the user manual supplied with the instrument.

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The QIAxcel Protein cartridge should be stored in the QX Cartridge Stand at room temperature (15–25°C) with the purge port sealed and covered with the QX Cartridge Stand Cover to protect it from light.

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When the QIAxcel Protein cartridge is used for the first time, or if a previously used cartridge has been stored inside the blister package for more than one month, a long purge should be performed to ensure optimal cartridge performance (for details, see step D8, page 40).

Procedure Cartridge preparation and setup 1. Add 10 ml QX Wash Buffer to both reservoirs of the QX Cartridge Stand and cover with 2 ml QX Mineral Oil. 2. Remove the QIAxcel Protein cartridge from its packaging and carefully wipe off any soft gel debris from the capillary tips using a soft, lint-free tissue. 3. Remove the purge cap tape from the back of the QIAxcel Protein cartridge. Clean any gel oozing out of the purge port with a soft tissue and then place the QIAxcel Protein cartridge in the QX Cartridge Stand. 4. Leave in upright position for 30 min.

Preparing the QIAxcel cartridge.

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QIAxcel Protein Handbook 04/2013

QX Buffer Tray preparation and setup We recommend using separate buffer trays for QIAxcel DNA, RNA, and Protein cartridges. 1. Add 8 ml QX Wash Buffer to the “WP” position of the QX Buffer Tray. 2. Fill the “WI” position of the QX Buffer Tray with 8 ml double distilled water. 3. Fill the “BUFFER” position of the QX Buffer Tray with 18 ml QX Pro Separation Buffer. 4. Add 2 ml QX Mineral Oil overlay to positions “WP” and “WI”, and add 4 ml QX Mineral Oil to the “BUFFER” position to prevent the buffer from evaporating. 5. Place the QX Buffer Tray carefully into the instrument. Note: The buffers in the buffer tray can be used until the expiration date indicated on the buffer bottle label. Only replace the buffers if spurious results occur (e.g., delay of migration or artificial signals occur over all channels).

Preparing the buffer tray and inserting the buffer tray into the buffer tray holder.

Intensity calibration of cartridge Every QIAxcel gel cartridge requires intensity calibration prior to sample analysis. The intensities of each capillary are normalized and a factor is applied for every subsequent run. This corrects for natural intensity reading variations between each capillary in the cartridge. The data for each cartridge intensity calibration are stored in a file named according to the cartridge and instrument identifiers (<cartridgeid>_<instrument-id>.xcc). This file is saved either in the default directory C:Documents and SettingsAll UsersApplication DataQIAGENQIAxcelScreenGelDataCartridgeCalibrationData or in a customized directory. If, for any reason, a different computer is used to the one on which the calibration file is saved, the calibration file should be transferred to the new computer. Otherwise, recalibration of the cartridge is required. Similarly, if the

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QIAxcel gel cartridge is used on a different QIAxcel instrument to the one it was calibrated on, another intensity calibration should be performed. 1. Load 15 µl QX Pro Calibration Marker into each well of a QX Color 0.2 ml 12-Tube Strip and insert the strip into the “MARKER2” position of the QX Buffer Tray. 2. Place the QIAxcel gel cartridge into the instrument as described in the instrument user manual. 3. Launch the calibration run by clicking the “Start calibration” button in the “Calibration” screen of the “Service” environment. 4. Once the calibration is complete, the calibration results are displayed next to the gel image or the electropherogram view. The result table shows the area, calibration factor, and the result (“Pass” or “Fail”) for each channel. Note: A successfully calibrated cartridge should have a normalized area (NA) calibrated range between 0.05–0.07. If one or more channels show no signals in the first run, refer to Appendix D, page 39. If one or more channels showed high background, refer to Section 8 of the instrument user manual. If calibration failed more than twice, call QIAGEN Technical Services. Recalibration using QIAxcel ScreenGel software To recalibrate a cartridge, repeat the procedure described above. The calibration results of the previous calibration procedure are discarded when recalibrating a cartridge. Note: It is possible to calibrate a cartridge for which no calibration runs remain. In this case, 3 of the remaining regular runs are used instead of 1 calibration run.

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Protocol: Preparation of Samples and Marker The protein samples require denaturing and labeling prior to sample separation and analysis with the QIAxcel or QIAxcel Advanced system. Important points before starting

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Allow all required reagents to thaw to room temperature (15–25°C) and vortex before use.

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We recommend working with 5 µl of sample and diluting it after the labeling reaction if required.

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For information on the QIAxcel or QIAxcel Advanced, please refer to the user manual supplied with the instrument.

Things to do before starting

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If working with a master mix, freshly prepare the master mix and add QX Pro Labeling Dye shortly before reaction start and as the last component of the mixture.

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In general, avoid protein sample buffers with high concentrations of salt (NaCl concentrations should not exceed 250 mM), since other ions influence sample injection, and ultimately the separation of proteins. In addition, high concentrations of primary amines should be avoided due to the binding properties of the fluorescent dye. However, Tris-buffers with a maximum concentration of 50 mM are acceptable.

Procedure 1. Pipet sample or QX Pro Marker 14–97 kDa, QX Pro Labeling Buffer, QX Pro Reduction Agent, and QX Pro Labeling Dye into a microreaction tube according to Table 1. Table 1. Pipetting scheme for one sample labeling reaction Reagent

Volume (µl)

Protein sample or QX Pro Marker 14–97 kDa

5

QX Pro Labeling Buffer

7.5

QX Pro Reduction Agent

1.5

QX Pro Labeling Dye

1

Final sample volume

15

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2. Optional: To label the sample using a master mix, prepare a master mix as described in Table 2 and add 5 µl sample/marker to 10 µl of master mix. Table 2. Preparation of a master mix for a labeling reaction Number of Volume of QX Volume of QX labeling Pro Labeling Pro Reduction reactions Buffer Agent

Volume of QX Final Pro Labeling volume Dye

12

97.5 µl

19.5 µl

13 µl

130 µl

24

195 µl

39 µl

26 µl

260 µl

96

750 µl

150 µl

100 µl

1000 µl

3. Vortex gently for a few seconds. Proceed with incubation immediately after mixing samples with the buffer, reagent, and dye. 4. Incubate the samples and QX Pro Marker 14–97 kDa for 20 min at 50°C and briefly centrifuge to collect any condensed liquid at the bottom of the tube. The color of the labeling reaction changes from blue to pink. The intensity of color change depends on the protein concentration in the mixture. 5. Add 50 µl QX Pro Labeling Buffer to QX Pro Marker 14–97 kDa. Protein concentrations in the samples may be also adapted after the labeling reaction by the addition of QX Pro Labeling Buffer, in case of high protein concentration. 6. Run and analyze the samples on the QIAxcel Advanced instrument. Samples should be processed within 8 h of labeling. If not processed immediately, ensure that the samples are not exposed to light. Note: If more than 3 strips are run, add one drop of QX Mineral Oil on top of the samples to avoid evaporation.

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Protocol: Starting a Sample Run Running protein samples on the QIAxcel Advanced instrument requires calibration of the cartridge prior to sample run and labeling of samples or marker shortly before separation. See protocols “System Preparation” (page 12) and “Preparation of Samples and Marker” (page 15). Important points before starting



To operate the QIAxcel or QIAxcel Advanced System, please refer to the user manual supplied with the instrument.

Procedure 1. Load 15 µl QX Pro Alignment Marker into each well of a QX 0.2 ml 12-Tube Strip. Add 1 drop of QX Mineral Oil to each well and insert the strip into the QX Buffer Tray in the “MARKER1” position. Note: QX Pro Alignment Marker should be replaced after 24 runs. 2. Place the labeled samples and marker into the instrument. The minimum final sample volume required for analysis is 10 µl. Less than 0.1 µl of the sample will be injected into the QIAxcel gel cartridge for analysis. 3. Start the electrophoresis run using the default or customized protein process profile. Note: To customize the process profile, see Section 6.3.2 of the QIAxcel Advanced User Manual.

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Figure 2. Starting a run in “Advanced” mode.

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Protocol: Analysis of Samples This section provides instructions on sample analysis after a run, in case the analysis option was not preselected in the process profile. There are 2 types of analyses - analysis of samples of low complexity and analysis of samples of high complexity. For all types of samples, first perform steps 1–6 below. Then, follow guidelines for the specific sample type. Important points before starting

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To operate the QIAxcel or QIAxcel Advanced System, please refer to the user manual supplied with the instrument.

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For detailed analysis instructions, see Section 6.4.8 of the QIAxcel Advanced User Manual.

Procedure Analysis of all types of samples 1. Switch to “Protein mode” if this is not already selected. 2. Load the samples you want to analyze using the “Experiment Explorer” in the “Analysis” environment. 3. If a size marker was run alongside samples, create a new reference marker as described on page 22. 4. Visualize the samples you want to analyze. 5. Select the samples to be analyzed. Open the “Analysis” parameters panel to specify the analysis properties. These depend on the content of the sample being analyzed. Therefore, follow either the section entitled “Analysis of samples of low complexity” (page 19) or to the section entitled or “Analysis of samples of high complexity” (page 20). Analyzing samples of low complexity This section describes the analysis of samples of low complexity (e.g., samples containing a single protein or a few proteins). 1. Select the “Default Protein” analysis profile. 2. In the reference marker section, check “Reference Marker Table” and select a previously created reference marker. 3. Click “Start Analysis” to start the analysis. 4. After the analysis has been completed, the single electropherogram views of the analyzed samples will show the size of the detected proteins in kilodaltons (kDa) above the apex of the corresponding peaks.

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5. Ensure that peak detection has worked as expected. The single electropherogram view is most suitable for checking peak detection. Note: As QIAxcel ScreenGel software is able to determine protein sizes in the range from 10 to 100 kDa, smaller or larger proteins are not annotated with a size value. Instead “n/a” is displayed above the corresponding peaks. Note: If artifact peaks occur or spurious results are achieved, check Appendix B, page 32, for tips and tricks. Analyzing samples of high complexity This section describes the analysis of samples of high complexity such as cell lysates. 1. Select the “Protein Lysate” analysis profile. 2. In the reference marker section, check “Reference Marker Table” and select a previously saved reference marker. 3. Click “Start Analysis” to start the analysis. 4. Ensure that the peak detection has worked as expected. The single electropherogram view is most suitable for checking peak detection. 5. As the analyzed sample is of high complexity, usually a lot of peaks are detected. Nevertheless, if there are system peaks of non-protein origin (artifacts) in the electropherogram, remove them using “Suspend Integration” as described in Appendix B, page 32. 6. If the baseline follows the signal, increase the baseline filter window. This is done by clicking on the parameter value and changing the window size. Then click “OK”. 7. Click “Start Analysis” again to start a new analysis according to this modified analysis profile. In some cases, it may be necessary to repeat the adaptations to the analysis profile. If the peak detection worked as expected with the modified analysis profile, save this analysis profile and try to use this new profile instead of the “Protein Lysate” analysis profile for future protein analyses. 8. Check the peak detection again in the single electropherogram view.

Additional information Relative concentration The column “Rel. Conc. [ng/µl]” displays the relative concentration for each detected peak. As for all peaks, the same reference peak of the size marker is used to calculate the concentration in relation to this reference. If this column is

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