ThermoFisher Scientific Invitrogen iBlot 3 User manual

For Research Use Only. Not for use in diagnostic procedures.
iBlot™3 Western Blot Transfer System
USER GUIDE
For dry electroblotting of proteins from mini and midi gels
Catalog Number IB31001
Publication Number MAN0028464
Revision A.0

Life Technologies Holdings Pte Ltd | Block 33 | Marsiling Industrial Estate Road 3 | #07-06, Singapore 739256
For descriptions of symbols on product labels or product documents, go to thermofisher.com/symbols-definition.
Revision history:MAN0028464 A.0 (English)
Revision Date Description
A.0 5 January 2023 New document for iBlot™3 Western Blot Transfer System.
The information in this guide is subject to change without notice.
DISCLAIMER: TO THE EXTENT ALLOWED BY LAW, THERMO FISHER SCIENTIFIC INC. AND/OR ITS AFFILIATE(S) WILL NOT BE
LIABLE FOR SPECIAL, INCIDENTAL, INDIRECT, PUNITIVE, MULTIPLE, OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR
ARISING FROM THIS DOCUMENT, INCLUDING YOUR USE OF IT.
Important Licensing Information: These products may be covered by one or more Limited Use Label Licenses. By use of these
products, you accept the terms and conditions of all applicable Limited Use Label Licenses.
TRADEMARKS: All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified.
©2023 Thermo Fisher Scientific Inc. All rights reserved.

Contents
■CHAPTER1Productinformation .................................................. 6
Product contents ................................................................ 6
iBlot™3 Western Blot Transfer Device contents ................................. 6
Before starting ............................................................. 6
Installing theinstrument ..................................................... 7
Service operation requirements ............................................... 8
Unpackinginstructions .......................................................... 8
Upon receiving theinstrument ................................................ 8
Unpacking the iBlot™3 Western Blot TransferDevice ............................ 8
iBlot™3 Western Blot TransferDevice .............................................. 8
Deviceviews ............................................................... 8
About the system ............................................................... 9
iBlot™3 Western Blot Transfer System ......................................... 9
Features ................................................................... 9
Systemcomponents ........................................................ 9
System overview ........................................................... 9
Description of parts ............................................................ 11
iBlot™3 Western Blot TransferDevice ........................................ 11
Blotting surface ........................................................... 11
Lids andcooling ........................................................... 12
Controlpanel .............................................................. 12
Blotting roller .............................................................. 12
Power cord and power adapters ............................................. 12
iBlot™3 Transfer Stacks .................................................... 12
Guidelines for iBlot™3 Transfer Stacks ....................................... 13
Topstack ................................................................. 14
Bottomstack .............................................................. 15
Transfer membrane ........................................................ 16
iBlot™Filter Paper .......................................................... 17
iBlot™3 Absorbent Pad ..................................................... 17
iBlot™3 Western Blot Transfer System User Guide 3

Operating the iBlot™3 Western Blot TransferDevice ................................ 18
First time usage of the iBlot™3 Western Blot TransferDevice .................... 18
Control panel of the iBlot™3 Gel TransferDevice .............................. 18
Set the date andtime ...................................................... 19
Write logs to a USB storagedevice .......................................... 19
Description ofmethods ......................................................... 20
Methods .................................................................. 20
Recommended running parameters .......................................... 20
■CHAPTER2Protein transfer protocol ........................................... 22
Experimental overview .......................................................... 22
Experimentaloutline ....................................................... 22
Generalguidelines ............................................................. 22
Recommended geltypes ................................................... 23
Using the iBlot™3 Western Blot Transfer System ................................... 23
Materialsneeded .......................................................... 23
Select amethod ........................................................... 24
Assemble the iBlot™3 Transfer Stack ......................................... 24
Performblotting ........................................................... 26
Disassemble the iBlot™3 Transfer Stack ...................................... 26
■CHAPTER3Custommethods ................................................... 28
Create custommethods ........................................................ 28
Create a newmethod ...................................................... 28
Edit existingmethods ...................................................... 30
Create custom methods from an existing template (up to 10 steps) .............. 31
■APPENDIXATroubleshooting .................................................... 32
Introduction ................................................................... 32
■APPENDIXBPost transferanalysis .............................................. 35
Post transferanalysis ........................................................... 35
■APPENDIXCOptimizingblotting ................................................ 36
Optimizingblotting ............................................................. 36
Contents
4iBlot™3 Western Blot Transfer System User Guide

■APPENDIXDMaintenance ....................................................... 37
Cleaning ...................................................................... 37
General maintenance ........................................................... 37
Replacing thefuse ............................................................. 37
Replacing electricalcontacts .................................................... 39
■APPENDIXEProductspecifications ............................................. 41
iBlot™3 Western Blot Transfer Devicespecifications ................................ 41
iBlot™3 Transfer Stackspecifications ............................................. 42
■APPENDIXFAccessory products ............................................... 43
iBlot™3 Transfer Stacks ......................................................... 43
Additional products ............................................................ 43
Precast gels and premadebuers ................................................ 44
■APPENDIXGSafety ............................................................... 45
Symbols on thisinstrument ..................................................... 45
Standard safetysymbols ................................................... 45
Control and connectionsymbols ............................................. 46
Safety information for instruments not manufactured by Thermo FisherScientific ...... 47
Instrumentsafety .............................................................. 47
Electricalsafety ........................................................... 47
Cleaning anddecontamination .............................................. 48
Instrument component and accessorydisposal ................................ 48
Safety and electromagnetic compatibility (EMC) standards .......................... 48
Symbols oninstrument ..................................................... 48
Safety standards .......................................................... 49
EMC standards ............................................................ 49
Environmental design standards ............................................. 50
Radio compliance standards ................................................ 50
Chemicalsafety ................................................................ 51
■APPENDIXHDocumentation and support ...................................... 52
Customer and technical support ................................................. 52
Limited product warranty ........................................................ 52
Contents
iBlot™3 Western Blot Transfer System User Guide 5

Product information
Product contents
iBlot™3 Western Blot Transfer Device contents
This manual is intended to provide instructions for the operation of the iBlot™3 Western Blot Transfer
Device (Cat. No.IB31001).
The contents of the iBlot™3 Western Blot Transfer Device are listed below. See “iBlot™3 Western Blot
Transfer Device specifications” on page41 for specifications and description of the iBlot™3 Western
Blot Transfer Device.
Component Quantity
iBlot™3 Western Blot Transfer Device 1
Blotting Roller 1
Power Cord 1
Before starting
Before you begin using this product, or any installation or service operation, please read the following
safety information. Attention to these warnings will help prevent personal injuries and damage to the
products.
It is your responsibility to use the product in an appropriate manner. This product is designed for use
solely in laboratory environments, and must not be used in any way that may cause personal injury or
property damage.
You are responsible if the product is used for any intention other than its designated purpose
or in disregard of Thermo Fisher Scientific instructions. Thermo Fisher Scientific shall assume no
responsibility for such use of the product.
The product is used for its designated purpose if it is used in accordance with its product
documentation and within its performance limits.
Using the product requires technical skills and a basic knowledge of English. It is therefore essential
that only skilled and specialized sta or thoroughly trained personnel with the required skills be allowed
to use the product.
Keep the basic safety instructions and the product documentation in a safe place and pass them on to
the subsequent users.
Applicable local or national safety regulations and rules for the prevention of accidents must be
observed in all work performed.
1
6iBlot™3 Western Blot Transfer System User Guide

The iBlot™3 Western Blot Transfer Device complies with the TUV Rhineland North America Inc. safety
requirements, part 15 of the FCC rules, and the European Community Safety requirements. Operation of
the iBlot™3 Western Blot Transfer Device is subject to the conditions described in this manual.
Operation of the iBlot™3 Western Blot Transfer Device is subject to the following conditions:
•Indoor use
•Altitude below 2,000 meters
•Temperature range: 4 to 30°C
•Maximum relative humidity: 80% (maximum relative humidity 80% for temperatures up to 31°C,
decreasing linearly to 50% relative humidity at 40°C)
•Installation categories (over voltage categories) II; Pollution degree 2
•Mains supply voltage fluctuations not to exceed 10% of the nominal voltage (100–240 V, 50/60 Hz,
6.3 A)
•Mains plug is a disconnect device and must be easily accessible.
•Do not attempt to open the iBlot™3 Western Blot Transfer Device. To honor the warranty, the iBlot™
3 Western Blot Transfer Device can only be opened and serviced by Thermo Fisher Scientific.
•The protection provided by the equipment may be impaired if the equipment is used in a manner
not specified by Thermo Fisher Scientific.
•The device must be connected to a mains socket outlet with protective earthing connections.
•Ventilation requirements: Room ventilation
•Do not use if device becomes cracked or broken in any area.
Installing the instrument
The product may be installed only under the conditions and in the positions specified by Thermo Fisher
Scientific.
Following are the required operating position and conditions:
•Do not place the product in an area where it will be subject to vibration.
•Do not place the product on surfaces, vehicles, cabinets or tables that for reasons of weight or
stability are unsuitable for this purpose.
•Do not place the product on heat-generating surface or near heat emitting devices such equipment
racks or heaters. Verify that there is sucient clearance between the product and any other system
that may exhaust warm air.
•The product's ventilation should not be obstructed. If proper ventilation is not provided it can result
in electric shock, fire and/or serious personal injury or death.
•The product is for indoor use only.
•Use only with suitably rated mains supply cord (having 3 conductors, min. 16 AWG or 1.5 mm2,
min. 300V, Harmonized Type for Europe and UL Listed/CSA Certified for North America, with
molded plug rated min. 10A).
•A tolerance of ±10% shall apply to the nominal input voltage and ±3% to the nominal frequency,
over voltage category 2.
•Maximum operating altitude 2,000 m asl. Maximum transport altitude 4,500 m asl.
Chapter1Product information
Product contents 1
iBlot™3 Western Blot Transfer System User Guide 7

Service operation requirements
In the event of an equipment malfunction, it is the responsibility of the customer to report the need for
service to Thermo Fisher Scientific or to one of the authorized agents. For service information, contact
Technical Support.
Servicing of this device is to be performed by trained service personnel only.
Unpacking instructions
Upon receiving the instrument
Examine the unit carefully for any damage incurred during transit. File any damage claims with the
carrier. The warranty does not cover in-transit damage.
Unpacking the iBlot™3 Western Blot Transfer Device
•Remove the roller from the Styrofoam packaging.
•Remove the device from the Styrofoam packaging.
•Remove the protective film from the touch screen.
•Remove blue protective tape from the electrode contact positions on the base.
iBlot™3 Western Blot Transfer Device
Device views
Figure1Front, rear, and top views of the iBlot™3 Western Blot Transfer Device
Chapter1Product information
Unpacking instructions
1
8iBlot™3 Western Blot Transfer System User Guide

About the system
iBlot™3 Western Blot Transfer System
The iBlot™3 Western Blot Transfer System consists of the iBlot™3 Western Blot Transfer Device and
associated iBlot™3 Transfer Stacks (sold separately). The iBlot™3 Western Blot Transfer Device has a
unique design, which, in conjunction with the patented gel matrix technology of the iBlot™3 Transfer
Stacks, results in a shortened distance between electrodes, high field strength, and high currents to
reduce transfer times when blotting proteins onto membranes.
Western blotting of proteins from midi- or mini-sized polyacrylamide gels onto nitrocellulose or PVDF
membranes can be performed with iBlot™3 Transfer Stacks in less than 10 minutes.
Features
•Pre-programmed methods for protein transfers from various gel types in 3–8 minutes
•Enhanced user safety with built-in safety features in the device
•User-friendly design with an integrated power supply to avoid inconsistencies associated with the
use of an external power supply
•Fast, reliable protein transfer with ready-to-use iBlot™3 Transfer Stacks with no need to prepare
buers or membranes
•Compatible for use with Novex™and NuPAGE™gel types and other gels
•Not compatible with E-PAGE™gels
System components
iBlot™3 Western Blot Transfer Device
The iBlot™3 Western Blot Transfer Device is a self-contained blotting unit with integrated power supply
used for fast dry blotting of proteins.
iBlot™3 Transfer Stacks
The iBlot™3 Transfer Stacks are disposable stacks that have integrated PVDF or nitrocellulose transfer
membranes to perform dry blotting of proteins. Each iBlot™3 Transfer Stack contains a copper
electrode and appropriate cathode and anode buers in the gel matrix to allow fast, reliable transfer of
proteins. See “iBlot™3 Transfer Stacks” on page43 for details.
System overview
The iBlot™3 Western Blot Transfer System is based on the dry blotting concept, utilizing the unique,
patented gel matrix technology developed for E‑Gel™gels and applied in iBlot™3 Transfer Stacks.
Each iBlot™3 Transfer Stack consists of a Bottom Stack and a Top Stack sandwiching a pre-run gel
and a nitrocellulose or PVDF membrane. The iBlot™3 Transfer Stack is assembled with the blotting
membrane on the anode side, and a pre-run gel on the cathode side.
Chapter1Product information
About the system 1
iBlot™3 Western Blot Transfer System User Guide 9

Schematic of iBlot™3 Transfer Stack showing the flow of current
9
+
_
Current flow
1
3
4
5
6
7
8
2
1Top felt with folded aluminum tab
2Copper cathode
3Cathode gel matrix
4Plastic divider
5Membrane (NC or PVDF)
6Improved anode gel matrix
7Copper anode
8Bottom felt
9Transfer tray
After the stack is assembled on the iBlot™3 Western Blot Transfer Device, and the appropriate
method is selected, the run is initiated. Transfer of proteins from the gel to the blotting membrane
is accomplished in approximately 3–10 minutes. The rapid transfer without the need for external power
supply or premade buers is possible due to the following features of the iBlot™3 Western Blot Transfer
System:
•The gel matrix of the Bottom Stack and Top Stack incorporates the appropriate anode and cathode
buers to act as ion reservoirs. This format eliminates the need for premade buers or soaked
membranes and minimizes handling that can lead to inconsistent performance.
•The copper anode does not generate oxygen gas as a result of water electrolysis, resulting in
increased transfer consistency. Conventional inert electrodes present in other blotting systems
result in oxygen generation, which can result in blotting distortion.
•The transfer stack gel matrix technology and reduced distance between the electrodes combined
with the integrated power supply allows the system to generate high field strength and increase
transfer speed.
Chapter1Product information
About the system
1
10 iBlot™3 Western Blot Transfer System User Guide

Description of parts
iBlot™3 Western Blot Transfer Device
The iBlot™3 Western Blot Transfer Device is a protein transfer device with an integrated power supply
capable of producing currents up to 6.3 amp, and supplying voltage up to 35 V. Four printed circuit
boards hold the electronic components required to process the systems logic unit, modify voltage and
currents for display, and power the blotting process. Pre-installed firmware controls the parameters
such as voltage, time, and cooling, and allows selection of Methods (see “Description of methods” on
page20 for details on each Method).
IMPORTANT! When installing the iBlot™3 Western Blot Transfer Device, make sure it is placed on a
level surface. Keep the area around the device clear to ensure proper ventilation of the unit. For your
safety: Position the device properly such that the Power switch and the AC inlet located at the rear of
the unit (“Device views” on page8) are easily accessible.
Figure2Device with lid open and detailed parts
Blotting surface
The blotting surface is the area where the iBlot™3 Transfer Stack containing the gels is placed to
perform blotting. Alignment guides are used for proper orientation of midi and mini transfer stacks.
Each station (Station 1 and Station 2) can accommodate one or two mini stacks, or one midi stack.
Chapter1Product information
Description of parts 1
iBlot™3 Western Blot Transfer System User Guide 11

Lids and cooling
The lids of the iBlot™3 Western Blot Transfer Device exert even pressure on the stack surface when the
lid is closed.
The lids contain a cooling system in the form of a heat sink connected to, and in thermal
communication, with the cathode plate. During the transfer run, there is a heat exchange between
the transfer stack and the cathode plate, and from the cathode plate back to the heat sink. The air
surrounding the fins of the heat sink facilitates continuous cooling of the stack and the extent of cooling
is controlled by the fan.
Control panel
The graphical Touch Screen User Interface is an LCD display allowing the user to select methods and
control the device by following menu prompts.
Blotting roller
The Blotting Roller is a plastic roller attached to a stainless steel handle (8.6cm wide). The Blotting
Roller is used to remove any air bubbles between the gel and blotting membrane during the assembly
of the stacks and gel.
Power cord and power adapters
The Power Cord connects to the iBlot™3 Western Blot Transfer Device on one end, and to a power
adapter (for plugging into an AC electrical outlet) on the other.
IMPORTANT! Be sure that the AC power switch is in the OFF position (see “Replacing the fuse” on
page37 for a detail view of the switch) before attaching the power cord. Attach the power cord to the
AC inlet of the device first, and then to the electrical outlet. Use only properly grounded AC outlets and
power cords.
Use the appropriate power cord and power adapter for your geographical region.
iBlot™3 Transfer Stacks
The iBlot™3 Transfer Stacks are used to transfer proteins from gels onto nitrocellulose or PVDF
membranes, and are available in Midi size for blotting one midi or two mini gels, and Mini size for
blotting one mini gel.
Chapter1Product information
Description of parts
1
12 iBlot™3 Western Blot Transfer System User Guide

Guidelines for iBlot™3 Transfer Stacks
•Store the iBlot™3 Transfer Stacks at room temperature. For best results, use the transfer stack
before the expiration date printed on the package for each stack.
•Do not remove transfer stacks from bottom plastic tray. The plastic tray is a central part of the
consumable. It maintains the current within the stacks and separates it from the rest of the device.
The plastic tray also contains any free liquid, allowing for easier clean-up.
•Discard the iBlot™3 Transfer Stack after every use. Do not reuse the iBlot™3 Transfer Stack
or any of its components.
•iBlot™3 Transfer Stacks are compatible only with the iBlot™3 Western Blot Transfer Device.
Use iBlot™3 Transfer Stacks only for their designated application.
Note: The maximum voltage and current of the output to the gel stacks is 35 VDC and 6.5 A.
The following iBlot™3 Transfer Stacks are available at thermofisher.com/iblot3 (see “iBlot™3 Transfer
Stacks” on page43 for ordering information).
Each iBlot™3 Transfer Stacks box comes with the following components: 10 iBlot™3 Transfer Stacks
(mini or midi), 10 iBlot™3 Absorbent Pads (mini or midi), and 10 iBlot™Filter Papers (mini or midi).
Product Transfer Membrane Cat. No.
iBlot™3 Transfer Stacks, Midi Nitrocellulose
PVDF
IB33001
IB34001
iBlot™3 Transfer Stacks, Mini Nitrocellulose
PVDF
IB33002
IB34002
Chapter1Product information
Description of parts 1
iBlot™3 Western Blot Transfer System User Guide 13

Top stack
The Top Stack is separated from the Bottom Stack by a white plastic divider and contains a copper
electrode and a transfer cathode gel layer. The transfer gel layer acts as an ion reservoir and is
composed of an optimized, proprietary gel composition.
3
1
2
1Copper electrode
2Cathode gel matrix
3Plastic divider
Chapter1Product information
Description of parts
1
14 iBlot™3 Western Blot Transfer System User Guide

Bottom stack
The Bottom Stack in the plastic tray contains an absorbent pad, a copper electrode, transfer anode gel
layer, and a nitrocellulose or PVDF membrane for protein transfer. The transfer gel layer acts as an ion
reservoir and is composed of an optimized, proprietary gel composition. The transparent plastic tray in
which the iBlot™3 Transfer Stack is packaged serves as the support for assembling the transfer stack
with the gel, and as a reservoir to contain any liquid generated during the blotting process.
The nitrocellulose and PVDF membranes do not require any pretreatment before use.
During the transfer, always use the Bottom Stack with the tray in the iBlot™3 Western Transfer
Device.
1
2
3
4
5
1Blotting membrane
2Improved anode gel matrix
3Copper electrode
4Absorbent pad
5Plastic tray
Chapter1Product information
Description of parts 1
iBlot™3 Western Blot Transfer System User Guide 15

Transfer membrane
The iBlot™3 Transfer Stacks are assembled with the transfer membrane and are available with:
•Nitrocellulose membrane (0.2µm)
The nitrocellulose membrane is composed of 100% pure nitrocellulose to provide high-quality
transfer. The membrane is compatible with commonly used detection methods such as staining,
immunodetection, fluorescence, or radiolabeling. The proteins bind to the membrane due to
hydrophobic and electrostatic interactions. The protein binding capacity is 209µg/cm2.
•PVDF membrane (0.2µm, low fluorescence)
The PVDF membrane has higher binding capacity than nitrocellulose. The PVDF membrane
is preactivated and ready for use (no need for additional pretreatment with alcohol).
The membrane is compatible with commonly used detection methods such as staining,
immunodetection, fluorescence, or radiolabeling. The proteins bind to the membrane due to
hydrophobic interactions. The protein binding capacity is 240µg/cm2.
Chapter1Product information
Description of parts
1
16 iBlot™3 Western Blot Transfer System User Guide

iBlot™Filter Paper
The iBlot™Filter Paper is supplied in two sizes for ecient blotting of mini or midi gels. Briefly wetted in
deionized water, the iBlot™Filter Paper is placed on top of the pre-run gel before placing the Top Stack
to protect the gel integrity during the blotting process.
Note: Failure to use the iBlot™Filter Paper during blotting of mini- or midi gels may result in high
currents exceeding the current limit leading to a “High Current Error” during the run.
iBlot™3 Absorbent Pad
The iBlot™3 Absorbent Pad absorbs any excess liquid formed during blotting and generates even
pressure on the stack assembly. The aluminum tab on each side of the pad acts as an electrical contact
between the assembled stack and the cathode plate.
Absorbent Pad (Midi)
Absorbent Pad (Mini)
Copper tab
Folded aluminum tab
When properly assembled, the aluminum tab of the iBlot™3 Absorbent Pad is placed away from the
copper tab and allows completion of the electrical circuit.
Discard the iBlot™3 Absorbent Pad after every use. Do not reuse the iBlot™3 Absorbent Pad.
Chapter1Product information
Description of parts 1
iBlot™3 Western Blot Transfer System User Guide 17

Operating the iBlot™3 Western Blot Transfer Device
First time usage of the iBlot™3 Western Blot Transfer Device
The first time the iBlot™3 Western Blot Transfer Device is turned on, you will need to perform the
following actions:
1. Accept the End User License agreement.
2. Configure Date & Time including Time Zone, Date/Format, and Time/Format by following the
screen prompts.
3. Change the Instrument Name if desired.
Control panel of the iBlot™3 Gel Transfer Device
The control panel is a touch screen display allowing the user to run the transfer method and access
device settings.
1. Press Settings. The Settings screen allows the user to perform the following actions:
About Instrument Instrument Settings Maintenance & Services
•About instrument
•End User License Agreement
(EULA)
•Check updates
•Instrument name
•Date & time
•Sleep mode
•Brightness
•Buzzer control
•Firmware update
•Restore factory settings
•Self verification test
•For factory use
Run Log Error Log Methods Management
•Method name, Station, Date
(up to 250 run logs)
•Export run log
•Date, Error description
•Export error log
•Import, Export, or Delete user's
method
•Change method parameters and
rename
2. Run the method.
•Run a preset template (default pre-programmed method).
•Run the method last used on the instrument.
•Run a custom method.
Chapter1Product information
Operating the iBlot™3 Western Blot Transfer Device
1
18 iBlot™3 Western Blot Transfer System User Guide

Set the date and time
1. Press Accept on the End User License Agreement screen to access the Date & Time screen.
2. Touch the Date & Time screen to modify the Time Zone.
3. Touch the fields to choose a date format, then press Enter.
4. Set the date display in Short or Long version. Press Done to return to Date & Time screen.
5. Touch the fields to choose a time format (12- or 24-hr format).
6. Touch Done to return to the Date & Time screen. Press Next to access the Instrument Name
screen.
7. If desired, input the personal instrument name using the keyboard and press Enter.
8. Press Done to continue to the Set Up Run screen.
Write logs to a USB storage device
A record of each run is kept by the iBlot™3 Western Blot Transfer Device with transfer parameters
(voltage, current, time) and temperature being tracked at 1-second intervals. This information can be
downloaded to a USB storage device in a .csv format.
1. Insert a USB storage device into the USB port (Type A) located on the right side of the device.
Note: On average, the time to detect the USB drive once inserted is less than 10 seconds. USB
detection times vary depending on the USB drive.
2. Touch Run log on the Settings screen and select a single or multiple methods. Press Export.
3. Remove the USB storage device when the "Run log has been exported successfully" message
displays.
Chapter1Product information
Operating the iBlot™3 Western Blot Transfer Device 1
iBlot™3 Western Blot Transfer System User Guide 19

Description of methods
Methods
The iBlot™3 Western Blot Transfer Device is pre-programmed with 3 transfer methods using dierent
combinations of voltage, time, and cooling.
Transfer Method Run Parameters
Broad Range (30–250 kDa) 25 V, 6 mins, Low Cooling
High Molecular Range (150–400 kDa) 30 V, 8 mins, No Cooling
Low Molecular Range (10–120 kDa) 25 V, 3 mins, Medium Cooling
•Broad Range – Recommended for standard gel types and optimized for transfer of proteins
between 30–250 kDa.
•High Molecular Range – Recommended for Tris-Acetate gels and optimized for transfer of proteins
between 150–400 kDa.
•Low Molecular Range – Recommended for standard gel types and Tricine gels and optimized for
transfer of proteins between 10–120 kDa.
Recommended running parameters
The Default Run Time is the default time setting for a selected method. The Run Time Limit is the
maximum recommended run time for a selected method.
Table1Additional recommended parameters for protein transfer with a molecular weight range of
30–250 kDa
Transfer Stack Voltage Transfer Time Cooling
Midi transfer stacks
(2 mini gels or 1 midi gel)
25–30 V 5–7 mins No cooling or Low cooling
Mini transfer stack
(one mini gel)
25 V 5–6 mins Low cooling or Medium cooling
Table2Additional recommended parameters for protein transfer with a molecular weight <30 kDa.
Cooling setting is gel dependent. No cooling or low cooling is recommended for Tricine gels, but
some optimization is required.
Transfer Stack Volts Transfer Time Cooling
Midi transfer stacks
(2 mini gels or 1 midi gel)
20–25 V 3–5 mins Low cooling or Medium cooling
Mini transfer stack
(one mini gel)
25 V 2–4 mins Low cooling or Medium cooling
Chapter1Product information
Description of methods
1
20 iBlot™3 Western Blot Transfer System User Guide