Bio-Rad PROTEAN i12 IEF System User Manual

PROTEAN® i12™ IEF System

Instruction Manual

Catalog #164-6000, 164-6001

Bio-Rad Technical Support

For help and technical advice, please contact the Bio-Rad Technical Support department. In the United States, the Technical Support department is open Monday–Friday, 5:00 AM–5:00 PM, Paciﬁc time.

Phone: 1-800-424-6723 Fax: 1-510-741-5802 Email: LSG_TechServ_US@bio-rad.com (for U.S. and international customers)

Online technical support and worldwide contact information are available at

www.consult.bio-rad.com.

Legal Notices

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Bio-Rad reserves the right to modify its products and services at any time. This instruction manual is subject to change without notice. Although prepared to ensure accuracy, Bio-Rad assumes no liability for errors, or for any damages resulting from the application or use of this information.

Coomassie is a trademark of BASF Aktiengesellschaft. Excel and Microsoft are trademarks of Microsoft Corporation.

PROTEAN i12 IEF System Instruction Manualii |

Table of Contents

Chapter 1 PROTEAN® i12™ IEF System 1

1.1 System Components 2

1.1.1 PROTEAN i12 IEF Cell 2

1.1.2 Acce ssories 3

1.2 Unpacking and Setup 5

1.3 Workflow 5

Chapter 2 Basic Operation 7

2.1 Setup 7

2.1.1 i12™ Focusing Tray and Electrode Assemblies 7

2.1.2 Connecting the Electrodes 8

2.2 IPG Strip Rehydration and Sample Application 9

2.2.1 IPG Strip Rehydration in the Rehydration/ Equilibration Tray Followed by IEF 9

2.2.2 IPG Strip Rehydration in the Focusing Tray Followed by IEF 13

2.3 Starting the Run 14

Chapter 3 Running a Protocol 15

3.1 Workflow 16

3.2 Screen Details 18

Chapter 4 Creating and Editing Protocols 23

4.1 Workflow 23

4.2 Screen Details 24

Chapter 7 Troubleshooting 35

Chapter 8 Cleaning and Maintenance 39

Appendix A Reagent and Sample Preparation 40

Appendix B Sample Loading Methods and Running Configurations 42

Appendix C IEF Protocols 44

Appendix D IEF for Peptide Fractionation Prior to LC-MS 50

Appendix E References 51

Appendix F Specifications 52

Appendix G Ordering Information 53

Chapter 5 Setting Defaults and Managing Folders 27

5.1 Setting Default Parameters (Settings) 27

5.2 Managing Files and Folders (Files) 28

5.2.1 Navigating the Files Structure 28

5.2.2 Copying Files 29

Chapter 6 Data Export and Analysis 31

6.1 File Types 31

6.2 Export to Microsoft Excel Software 31

6.3 Export to PROTEAN i12 Reporter (www.i12Reporter.com) 32

PROTEAN i12 IEF System Instruction Manual | iii

Preface

Safety and Regulatory Compliance

This instrument has been certiﬁed to meet all applicable requirements of the EN61010-1 electrical equipment for measurement, control, and laboratory use standard and the class A standards for Electromagnetic Emissions, intended for laboratory equipment applications. It uses high output voltages that are electrically isolated from earth ground to minimize the risk of electrical shock to the user.

This product has also been tested to the requirements of CAN/CSA-C22.2 No. 61010-1, second edition, including Amendment 1, or a later version of the same standard incorporating the same level of testing requirements.

Certiﬁed products are safe to use when operated in accordance with the instruction manual.

Instrument Safety Warnings

This instrument should not be modiﬁed or altered in any way. Alteration of this instrument voids the warranty and safety certiﬁcation and creates a potential safety hazard.

This instrument is intended for laboratory use only. Bio-Rad Laboratories is not responsible for any injury or damage caused by the use of this instrument for purposes other than those for which it is intended, or by modiﬁcations of the instrument not performed by Bio-Rad Laboratories or an authorized agent. Follow the safety speciﬁcations listed in this section and throughout this manual. Use only the power cord supplied with the instrument, making sure to choose the plug adaptor that corresponds to the electrical outlets in your region.

The following guidelines should be observed and followed:

■

To ensure adequate cooling of the PROTEAN® i12™ IEF cell, be sure there is ≥6 cm clearance around the unit. Do not block the fan vents

■

Do not use cloth or absorbent pads underneath the unit. These or other loose items may be pulled into the fan intake, causing damage to the unit due to overheating and voiding the warranty

■

Connect the cell to a three-prong, grounded AC outlet using the three-prong AC power cord provided

■

Do not operate the instrument in extreme humidity (>90%) or where condensation can short the internal electrical circuits of the cell. The PROTEAN i12 IEF cell has passed tests for operation at 10–31ºC, 0–90% relative humidity (noncondensing). Operating the cell outside these conditions voids the warranty

■

Disconnect power to the PROTEAN i12 IEF cell before servicing. No user-serviceable parts are inside the instrument. Contact Bio-Rad service personnel for service

■

Emissions from this product may interfere with some sensitive appliances when placed nearby or on the same circuit as those appliances. Take appropriate measures to avoid interference

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PROTEAN i12 IEF System Instruction Manualiv |

PROTEAN® i12™ IEF

System

The PROTEAN i12 IEF system (Figure 1.1) is used for isoelectric focusing (IEF) on immobilized pH gradient (IPG) strips for the ﬁrst dimension of two-dimensional (2-D) electrophoretic protein analysis. The PROTEAN i12 IEF cell can run 1–12 IPG strips in 7, 11, 13, 17, 18, and 24 cm focusing trays. Each channel in the i12™ focusing tray is powered by its own power supply, enabling precise control over each IPG strip. This makes it possible to run different sample types, different gradients, and multiple protocols all at the same time.

The i12 focusing trays and electrode assemblies accommodate all possible gel conﬁgurations (gel-side up or down, with or without electrode wicks). It also allows sample loading either by inclusion in the rehydration solution (in-gel loading) or with sample cups (sample cup loading).

The cell is fully programmable from the user interface; connection to an external computer is not required. Each protocol can contain up to ten steps in which voltage, manner of voltage ramping, current, and duration (hr or V-hr) are deﬁned.

Fig. 1.1. PROTEAN i12 IEF system. The system includes the PROTEAN i12 IEF cell and numerous accessories.

PROTEAN i12 IEF System Instruction Manual | 1

Chapter 1 PROTEAN i12 IEF System

Preprogrammed protocols stored in the internal memory serve as a convenient starting point for developing optimized, sample-speciﬁc IEF conditions. A USB ﬂash drive can also be used as an alternative storage location or method of transfer for protocols and run data ﬁles.

PROTEAN i12 Reporter, a web-based application (www.i12reporter.com), is also available for uploading run data ﬁles, viewing electronic proﬁles for individual lanes, and comparing sample proﬁles from different runs. The application can be used to generate reports, print graphs, and create protocols. Protocols created with the application can be transferred to the PROTEAN i12 IEF cell using a USB ﬂash drive.

1.1 System Components

The PROTEAN i12 IEF system comprises the PROTEAN i12 IEF cell, electrode assemblies, i12 focusing trays, and other accessories that make IEF and system maintenance possible.

1.1.1 PROTEAN i12 IEF Cell

The PROTEAN i12 IEF cell (Figure 1.2, Table

1.1) contains 12 individual power supplies, each dedicated to a single IPG strip. This individual power control for each lane allows use of IPG strips with different pH gradients and sample types, concentrations, and conductivities in a single run. It also allows programming of different protocols.

The touch-screen user interface is used to operate the cell, retrieve preprogrammed and user-deﬁned protocols, create new or edit saved protocols, and access the internal memory for ﬁle management. New protocols, sample details, and run data are stored in the internal memory or on an external USB ﬂash drive.

User interface

Stylus storage

Opaque lid

Safety lid

Opaque lid

USB port

Power switch

Safety lid

Table 1.1. PROTEAN i12 IEF cell components.

Component Description

User interface Controls the PROTEAN i12 cell; touch screen operated by hand, stylus, or mouse

Opaque lid Protects light-sensitive labels from photobleaching

Safety lid Safety interlock

Peltier platform Holds one focusing tray and maintains temperature during the run

USB ports 4 USB ports (1 USB-A in front, 2 USB-A and 1 USB-B in back) for connection to a USB mouse and USB ﬂash drive(s)

Power switch For powering the cell on and off

Stylus storage Slot for storage of stylus

Internal memory System hardware; includes preprogrammed protocols and can be used to store user- deﬁned protocols and data ﬁles

Leveling feet Used to level the cell if needed

2 | PROTEAN i12 IEF System Instruction Manual2 |

Power switch

Fig. 1.2. PROTEAN i12 cell. A, Front view with all lids closed; B, Front view showing the safety lid closed and opaque lid open; C, Back view.

USB ports

Chapter 1 PROTEAN IEF System

1.1.2 Accessories

The PROTEAN i12 IEF system includes the accessories listed in Table 1.2 and shown in Figure 1.3.

Table 1.2. Accessories for the PROTEAN i12 system. Accessories not included may be purchased separately (see Appendix G, Ordering Information).

Component Quantity Description

Included with the PROTEAN i12 IEF System

i12 focusing trays 1 each For IEF (and rehydration) of 1–12 IPG strips; each tray includes 2 IPG with IPG strip retainers (7, 11, and 17 cm) strip retainers to maintain contact between the IPG strip and electrodes for IEF with the gel-side down conﬁguration

Electrode assemblies 1 set each Positive (+) and negative (-) electrode assemblies; provide contact between each IPG strip and its power supply

i12 rehydration/equilibration trays 25 each Disposable trays for passive rehydration and equilibration of IPG (7, 11, and 17 cm) strips

Electrode wicks 100 gel-side up Collect salts and other charged impurities as well as proteins with 500 gel-side down isoelectric points (pI) outside the pH range of the IPG strip (use is recommended)

ReadyStrip™ IPG strips 12 each Medium for isoelectric separation of proteins pH 3–10 (7, 11, and 17 cm)

Mineral oil 500 ml For overlay of IPG strips to prevent dehydration

Forceps 2 pair For manipulation of IPG strips

Cleaning brushes Set of 2 For cleaning the focusing tray and electrode assemblies

Cleaning concentrate 1 L For cleaning the focusing tray and electrode assemblies

USB ﬂash drive 2 Memory data storage device integrated with a USB interface; for storage and transfer of data from the PROTEAN i12 IEF cell

Stylus 3 Used to manipulate touch screen (user interface)

Leveling bubble 1 Indicates whether the IEF cell is level

Power cord 1 Connects the IEF cell to a power source

ReadyPrep™ rehydration buffer 10 ml For rehydration and sample loading of IPG strips

Not Included with the PROTEAN i12 IEF System

i12 focusing trays (13, 18, and 24 cm) For IEF (and rehydration) of 1–12 IPG strips; each tray includes 2 IPG strip retainers

i12 rehydration/equilibration trays (13, 18, and 24 cm) Disposable trays for passive rehydration and equilibration of IPG strips

Sample cup holder and sample cups For cup loading of samples

Cleaning concentrate

i12 rehydration/equlibration trays

ReadyStrip IPG strips

Fig. 1.3. PROTEAN i12 accessories.

Mineral oil

IPG strip retainers

Leveling bubble

USB flash drive

Electrode wicks

i12 focusing trays

ReadyPrep rehydration buffer

Electrode assemblies

Forceps

Styluses

Cleaning brushes

| 3PROTEAN i12 IEF System Instruction Manual | 3

Chapter 1 PROTEAN i12 IEF System

i12 Focusing Tray

Channels in the i12 focusing tray (Figure 1.4) hold IPG strips for IEF. Each focusing tray accommodates up to 12 IPG strips. Separate trays are available for 7, 11, 13, 17, 18, and 24 cm IPG strips. For ﬂexibility, the PROTEAN i12 focusing tray and electrode assemblies accommodate all run conﬁgurations: gel-side down or up, with in-gel sample loading or sample cup loading. Each focusing tray holds one negative (-) and one positive (+) electrode assembly. Focusing trays can also be used for rehydration of IPG strips.

Electrode Assemblies

The electrode assemblies (Figure 1.4) each include 12 individual sets of negative and positive electrodes. The assemblies attach to the focusing tray to provide contact between each IPG strip and its power supply. They accommodate all focusing tray sizes.

Positive (+) electrode assembly

Release clip with green tab

Orientation pin

Release clip with green tab

Focusing tray

Tray positioning stop

Fig. 1.4. PROTEAN i12 focusing tray and electrode assemblies.

IPG strip positioning stop

Channel

Release clip with green tab

Negative (-) electrode assembly

4 | PROTEAN i12 IEF System Instruction Manual4 |

Chapter 1 PROTEAN IEF System

1.2 Unpacking and Setup

1. Carefully inspect the shipping container for any damage that may have occurred during shipping. Severe damage to a container may indicate damage to its contents. If you suspect damage to the contents, immediately ﬁle a claim with the carrier in accordance with their instructions before contacting Bio-Rad Laboratories.

2. Open the shipping carton and lift the content out of its packing. Inspect the instrument for external damage. If any part is missing or damaged, contact Bio-Rad Laboratories immediately.

3. Place the PROTEAN i12 IEF cell on a ﬁrm, ﬂat surface.

a. Position the cell so that there is access to the USB ports (back panel) and power switch (right panel).

b. To ensure adequate cooling, be sure that there is ≥6 cm clearance around the unit. DO NOT block the

fan vents.

c. Make sure the system is on a level surface. Place the leveling bubble in the center of the cooling

platform and adjust the instrument leveling feet as needed.

4. Connect the cell to a three-prong, grounded AC outlet using the power cord provided with the cell.

Do not place cloth or absorbent pads underneath the instrument. These or other loose items may be pulled into the fan intake, causing damage to the unit due to overheating and voiding the warranty.

1.3 Workflow

Figure 1.5 summarizes the workﬂow described in this manual. For best results, use the reagents and protocols available in the ReadyPrep 2-D starter kit (catalog #163-2105) to familiarize yourself with the 2-D process and operation of the PROTEAN i12 IEF cell. For more details about performing 2-D electrophoresis, please refer to bulletin 2651, 2-D Electrophoresis for Proteomics: A Methods and Product Manual.

Fig. 1.5. IEF workflow overview.

| 5PROTEAN i12 IEF System Instruction Manual | 5

6 | PROTEAN i12 IEF System Instruction Manual6 |

Basic Operation

2.1 Setup

For best results, use the reagents and protocols available in the ReadyPrep™ 2-D starter kit (catalog #163-2105) to familiarize yourself with the 2-D process and operation of the PROTEAN® i12™ IEF cell.

2.1.1 i12™ Focusing Tray and Electrode Assemblies

The PROTEAN i12 electrode assemblies have 12 electrodes that fit the 12 channels in the i12 focusing tray. Each electrode accommodates the use of electrode wicks and both the gel side-up and gel side-down IEF configurations: a bridge on each electrode fits into the recessed area of the focusing tray to create the flat surface required for the gel-side down configuration, and the electrodes are spring-loaded, which allows them to exert a gentle downward pressure for the gel-side up configuration. Several features of the electrode assemblies ensure their correct and complete attachment to the focusing tray (Figure 2.1):

■

Square and round orientation pins in the electrode assemblies ensure correct positioning of the cathode and anode in the focusing tray

■

Release clips secure the electrode assemblies onto the focusing tray

■

Green tabs on the release clips help push the electrode assemblies into place

To place the electrode assemblies in the i12 focusing tray:

1. Grasp the electrode assembly by the release clips and position the orientation pins as shown in Figure 2.1.

2. Push down on the green tabs until the locks click into place on the walls of the focusing tray. With the gel-side down configuration, make sure that each electrode is properly seated in the recessed area of the focusing tray.

To remove electrode assemblies from the focusing tray, grasp the green tabs

on the release clips and gently squeeze inward.

PROTEAN i12 IEF System Instruction Manual | 7

Chapter 2 Basic Operation

Positive (+) electrode assembly

Release clip with green tab

Orientation

Release clip with green tab

IPG strip positioning stop

Channel

Focusing tray

Fig. 2.1. Placement of an electrode assembly onto the i12 focusing tray.

2.1.2 Connecting the Electrodes

1. Position the assembly on the Peltier platform. Use the positioning guides and stops on the platform and focusing tray as guides (Figure 2.2, see inset).

2. Slide the assembly toward the positive (+) end until the positive (+) electrode assembly is completely inserted. When the the tray positioning stop reaches the positioning guide on the platform, the assembly is seated correctly (Figure 2.2).

3. Make sure the negative pin on the negative (-) electrode is in direct contact with the metal gounding strip on the instrument (Figure 2.2).

Platform

positioning guide

Tray positioning

stop

Tray positioning

guide

Tray positioning

stop

Platform

positioning guide

Instrument

platform

Positioning the tray

Metal grounding strip

Negative pin

Fig. 2.2. Connecting the electrodes.

PROTEAN i12 IEF System Instruction Manual8 |

Chapter 2 Basic Operation

2.2 IPG Strip Rehydration and Sample Application

The choice of rehydration and sample loading method (in-gel sample loading or cup loading) and IPG strip conﬁguration (gel-side down or gel-side up) dictates the workﬂow for the run. The ﬁrst step is to rehydrate the IPG strips in rehydration solution, with or without sample (Table 2.1).

IPG strips can be rehydrated in either the i12 rehydration/equilibration trays or the i12 focusing tray:

■

For rehydration in rehydration/equilibration trays, see Section 2.2.1

■

For rehydration in the focusing tray, see Section 2.2.2. Rehydration in the focusing tray with in-gel sample application can be programmed as part of the IEF run

See Appendix B, Sample Loading Methods and Running Conﬁgurations, for guidelines for selecting the sample loading method and IPG strip conﬁguration.

Table 2.1. Rehydration volumes, sample loads, and mineral oil volumes. The values listed are recommendations. Optimum sample load depends on sample type. See Appendix A, Reagent and Sample Preparation, for more details.

IPG Strip Length 7 cm 11 cm 17 cm 18 cm 24 cm

Rehydration Solution 125 μl 200 μl 300 μl 315 μl 450 μl

Protein Load

Coomassie (Brilliant) Blue 50–100 µg 100–200 µg 200–400 µg 200–400 µg 400–800 µg Fluorescent stains 5–100 µg 20–200 µg 50–400 µg 50–400 µg 80–800 µg Silver stains 5–20 µg 20–50 µg 50–80 µg 50–80 µg 80–150 µg

Mineral Oil 4 ml 5 ml 7 ml 7 ml 9 ml

2.2.1 IPG Strip Rehydration in the Rehydration/Equilibration Tray Followed by IEF

Rehydration IEF

For the rehydration step:

1. Pipet the rehydration solution (with or without sample, see Table 2.1 for volumes and protein loads) along the center of the channel(s) of the i12 rehydration/equilibration tray. Take care to not introduce air bubbles when expelling the solution.

2. Using forceps, remove the cover sheet from the IPG strip, then gently place the IPG strip gel-side down onto the solution in the channel. Move the IPG strip back and forth slightly to ensure that the solution is distributed along the length of the IPG strips. Take care to not trap air bubbles beneath the IPG strip.

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Chapter 2 Basic Operation

3. Overlay each IPG strip with mineral oil to prevent evaporation and precipitation of urea during rehydration (see Table 2.1 for recommended volumes). Apply the mineral oil to both ends of the channel and allow it to flow toward the middle of the channel. IPG strips can be left to rehydrate for up to 1 hr before adding the mineral oil.

4. Cover the tray and leave it on a level bench overnight (12–18 hr) for complete rehydration.

5. Transfer the rehydrated IPG strips to the focusing tray for IEF (see below).

IEF with Gel-Side Up

For IEF of IPG strips that were rehydrated in the presence of sample (in-gel loading):

1. Using forceps, remove the IPG strips from the rehydration tray, remove excess mineral oil, and place the rehydrated IPG strips gel-side up in the channels of the focusing tray (Figure 2.3, A). Position the positive (+) ends of the IPG strips against the positioning stops in each channel.

2. (Recommended) Wet the gel-side up wicks (notched) with distilled or deionized water and blot off excess water. Use two wicks per IPG strip: place a wick at each end of each IPG strip (Figure 2.3, B).

3. Position the electrode assemblies in the focusing tray and press down on the green tabs to snap the electrode assemblies into place (Figure 2.3, C). Place the focusing tray with the rehydrated IPG strips on the Peltier platform and connect the electrodes to the instrument (see Section 2.1, Setup).

4. Overlay each IPG strip with mineral oil (see Table 2.1 for recommended volumes).

5. Select or program the protocol(s) and start the run (see Section 2.3, Starting the Run).

Place IPG strips gel-side up in channel of focusing tray.

(Recommended) Place wet electrode

wicks on both ends of the IPG strip.

IPG strip positioning stop

Place electrode assemblies in

the focusing tray. Press down on the green tabs to snap the assemblies into place.

Fig. 2.3. Placement of an IPG strip gel-side up into the i12 focusing tray.

PROTEAN i12 IEF System Instruction Manual10 |

Chapter 2 Basic Operation

IEF with Gel-Side Down

For IEF of IPG strips that were rehydrated in the presence of sample (in-gel sample loading):

1. Position the electrode assemblies in the focusing tray and press down on the green tabs to snap the electrode assemblies into place (Figure 2.4, A; see Section 2.1.1, i12 Focusing Tray and Electrode Assemblies).

2. (Recommended) Wet the rectangular (gel-side down) wicks with distilled or deionized water and blot off excess water. Use two wicks per IPG strip: place a wick on top of each electrode (Figure 2.4, B).

3. Using forceps, place the rehydrated IPG strips gel-side down in the channels of the focusing tray (Figure

2.4, C). Position the positive (+) ends of the IPG strips against the positioning stops in each channel.

4. Place the focusing tray on the Peltier platform and connect the electrodes to the instrument (see Section

2.1.2, Connecting the Electrodes).

5. Overlay each IPG strip with mineral oil (see Table 2.1 for recommended volumes).

6. Place the IPG strip retainers on top of the IPG strips at both the positive and the negative ends (Figure

2.4, D). Without IPG strip retainers in place, gases formed during electrolysis may lift IPG strips off the electrodes, interrupting electrical contact.

7. Select or program the protocol(s) and start the run (see Section 2.3, Starting the Run).

To avoid movement of the IPG strip retainers, which can damage the IPG strips, place them into the focusing tray after the focusing tray has been secured on the Peltier platform.

(Recommended) Place

wet electrode wicks in the channels of the focusing tray.

Place both electrode assemblies in the focusing tray. Press down

on the green tabs to snap the assemblies into place.

Place the focusing tray onto the Peltier platform,

connect the electrodes, and then place 2 IPG strip retainers on top of the IPG strips.

Place IPG strips gel-side down in the channels of the

focusing tray.

IPG strip positioning stop

Fig. 2.4. Placement of an IPG strip gel-side down into the i12 focusing tray.

| 11PROTEAN i12 IEF System Instruction Manual | 11

Chapter 2 Basic Operation

Cup Loading (IEF with Gel-Side Up)

Sample cups (catalog #164-6020) offer an alternative method of sample loading. Their use can improve resolution, especially at extreme pH ranges (see Appendix B, Sample Loading Methods and Running Conﬁgurations, for guidelines on when to use cup loading). The PROTEAN i12 sample cup assembly consists of a sample cup holder that holds 1–12 disposable sample cups.

1. Using forceps, place the rehydrated IPG strips gel-side up in the channels of the focusing tray (Figure 2.3, A). Position the positive (+) end of the IPG strips against the positioning stops in each channel.

2. (Recommended) Wet the gel-side up electrode wicks (notched) with deionized water and blot off excess water. Use two wicks per IPG strip: place a wick at each end of each IPG strip (Figure 2.3, B).

3. Position the electrode assemblies in the focusing tray and press down on the green tabs to snap the electrode assemblies into place (Figure 2.3, C). Place the focusing tray on the Peltier platform and connect the electrodes to the instrument (see Section 2.1.2, Connecting the Electrodes).

4. Prepare the sample cup assembly by placing the sample cups into the slots of the sample cup holder corresponding to the channel with the rehydrated IPG strip (Figure 2.5).

5. Clamp the sample cup assembly onto the edges of the focusing tray, on top of the IPG strips and next to either electrode (Figure 2.5). (Placement depends on the pH gradient and the sample. See Appendix B, Sample Loading Methods and Running Conﬁgurations.)

6. Load 25–250 µl sample into the sample cups (larger volumes of dilute samples may be loaded, up to 400 µl). Overlay both the sample in the sample cup and the IPG strip with mineral oil.

7. Select or program the protocol(s) and start the run (see Section 2.3, Starting the Run).

Two ﬂexible arms on the sample cup holder apply gentle pressure onto the sample cup to ensure a complete seal between the IPG strip and the sample cup.

Once the sample cup holder is positioned, any horizontal movement will damage the IPG strips.

Sample cup holder

Sample cup

Insert sample cup at an angle

12 |

Fig. 2.5. Placement of the sample cup assembly onto the focusing tray.

PROTEAN i12 IEF System Instruction Manual

Chapter 2 Basic Operation

2.2.2 IPG Strip Rehydration in the Focusing Tray Followed by IEF

Rehydration can be programmed as a part of the run, followed by the IEF protocols. Alternatively, the IPG strips can be rehydrated independently and the protocol(s) started when most convenient.

Rehydration IEF

1. Position the electrode assemblies in the focusing tray as described in Section 2.1.1.

2. Pipet the rehydration solution containing the protein sample along the center of the channel(s) of the focusing tray (see Table 2.1 for recommended volumes and protein loads). Do not introduce air bubbles when expelling the solution.

3. Using forceps, remove the cover sheet from the IPG strip, then gently place the IPG strip gel-side down onto the sample in the channel of the tray. To ensure even rehydration, move the IPG strips back and forth slightly to distribute the solution along the lengths of the IPG strips. Check that no bubbles are trapped beneath the IPG strip and that some rehydration solution extends beyond the electrode contacts.

4. Place the focusing tray with the IPG strips on the Peltier platform and connect the electrodes to the instrument (see Section 2.1.2).

5. Immediately overlay each IPG strip with mineral oil to prevent evaporation and precipitation of urea during rehydration. Apply the mineral oil to both ends of the channel and allow it to flow toward the middle of the channel. See Table 2.1 for recommended volumes of mineral oil.

6. Position the IPG strip retainers on top of the IPG strips at both the anode and the cathode to maintain electrical contact with the IPG strips during IEF. Without the IPG strip retainers, electrolysis gasses may lift IPG strips off of the electrodes, interrupting electrical contact.

Place IPG strip retainers after the focusing tray has been placed into the instrument to avoid movement of the strip retainers.

7. Rehydration in the focusing tray with in-gel sample application can be programmed as a part of the IEF run or be performed separately. To program rehydration as part of the run:

a. Select or program the protocol(s) for the lanes containing IPG strips (see Section 3.1).

b. Program the global rehydration conditions in the Run Settings screen (see Section 3.2). If electrode

wicks are used, include a pause to insert electrode wicks when the rehydration step is completed.

c. Start the run (see Section 2.3, Starting the Run).

For rehydration not programmed as part of the run, leave the tray on the Peltier platform or on a level bench overnight (12–18 hr) for complete rehydration.

Global rehydration conditions are applied to all IPG strips in a run.

| 13PROTEAN i12 IEF System Instruction Manual | 13

Chapter 2 Basic Operation

2.3 Starting the Run

Turn on the PROTEAN i12 IEF cell by pressing the power switch on the right side of the instrument. A self-diagnostic program runs for approximately 10 sec. On the user interface, a message reads Self Test in Progress. If a component fails, the diagnostic program stops, and an error message appears (see Chapter 7, Troubleshooting).

Once the cell is powered on, the Main screen appears (Figure 2.6). Start the run by selecting one of the options described in Figure 2.6.

Edit an existing protocol (Chapter 4)

Run an existing protocol (Chapter 3)

Manage files and folders (Chapter 5)

Fig. 2.6. PROTEAN i12 IEF cell Main screen.

Create a new protocol with up to 10 steps in the Edit Protocol screen (Chapter 4)

Set default parameters and customize screens (Chapter 5)

View onboard help

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