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2011 LSG GLOBAL MARCOM


Electrophoresis and Blotting

Trans-Blot Turbo™ Transfer System Ž

Getting to the Finish Line Faster


The Trans-Blot Turbo™ is a fast, efficient, and reproducible transfer system for transferring proteins from gels to membranes in as little as 3 minutes. ®

Turbo Transfers

Intuitive Interface

System Flexibility

High Throughput Ready-to-use Prepacked Consumables

TURBO PACK TURBO PACK TURBO PACK

Superior Transfer Efficiency

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www.transblotturbo.com

Getting to the Finish Line Faster!

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Bio-Rad introduces the Trans-Blot Turbo system — the next innovation in protein transfer. The Trans-Blot Turbo system reduces transfer protocols for gels to as little as 3 minutes, while maintaining high efficiency, throughput, and the flexibility to run turbo or traditional semi-dry protocols. Turbo Transfers with Trans-Blot Turbo Transfer Packs ■■ 3-minute protocol — A single Mini-PROTEAN ® TGX™ gel (for proteins with MW 5–150 kD) can be transferred in as little as 3 min ■■

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7-minute protocol — Up to 4 mini or 2 midi gels with mixed-molecular weight proteins (MW 5–150 kD) can be efficiently transferred in 7 min 10-minute protocol — Up to 4 mini or 2 midi gels with high-molecular weight proteins (MW 25–300+ kD) can be efficiently transferred in 10 min

.

3 min transfer

7 min transfer

10 min transfer

Protein transferred using different protocols. E. coli lysate (6 µg) was diluted two-fold. Samples were separated with Mini-PROTEAN TGX gels, transferred with the Trans-Blot Turbo system, stained with SYPRO Ruby and imaged on a VersaDoc™ 4000 MP system. Standards in lane 1 are Precision Plus Protein™ Unstained, with a top band of 250 kD MW.

Superior Transfer Efficiency ■■ Higher sensitivity and better transfer efficiency is seen with the Trans-Blot Turbo system in comparison to other blotting techniques. This data set demonstrates the successful transfer of the 1.25 ng protein band only with the Trans-Blot Turbo system. A. Trans-Blot Turbo system 10 ng

5 ng

C. Semi-dry blot

2.5 ng

B. Tank blot 1.25 ng

10 ng

5 ng

2.5 ng

1.25 ng

D. iBlot system

Superior transfer efficiency. Serial dilutions of transferrin were separated on a Criterion™ TGX™ 4–20% gel and transferred using four different blotting techniques. A, Trans-Blot Turbo system (25 V for 7 min); B, Tank blotting (100 V for 30 min); C, Semi-Dry (25 V for 30 min); D, iBlot (P3 for 7 min).

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www.transblotturbo.com

Throughput and Modularity ■■ High Throughput — Up to 4 mini or 2 midi gels can be transferred simultaneously, doubling the throughput of our nearest competitor ■■

Modular — Assemble and run transfers independently with the two cassettes. A single unit and multiple cassettes can be purchased to satisfy a whole lab’s blotting requirements

System Flexibility ■■ The Trans-Blot Turbo system accommodates both traditional semi-dry as well as rapid transfers

Throughput Comparison based on Transfer Methodology Tank 2

# of Mini Blots Transfer Time

Semi-dry

30 min +

4 30 min +

Current Method

Trans-Blot Turbo transfer

Intuitive Interface ■■ Provides customer confidence in protocol selection and execution. Select from optimized preloaded protocols or customize and save/recall up to 25 user-defined transfer protocols

5

2

4

7–10 min

3–10 min*

System Flexibility Transfer Efficiency

Throughput

Speed

Semi-dry transfer

Ready-to-use Prepacked Consumables ■■ Convenient, Green Transfer Packs — Ready-to-use TransBlot Turbo transfer packs eliminate extra membrane, filter paper, and buffer preparation. Setup time is reduced to one minute from the opening of the gel cassette to the start of the transfer. Environmentally friendly packaging is recyclable and generates minimal waste; proprietary buffer contains no methanol

Trans-Blot Turbo system

* Transfer times are optimized for specific molecular weight ranges

Tank transfer

TURBO PACK TURBO PACK TURBO PACK

iBlot system

● ●


Universal Rapid Transfer The Trans-Blot Turbo system was developed to deliver the most uniform transfer for all proteins regardless of molecular weight, post-translational modifications, or protein pI ■■

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6x stronger signal intensity — Signal intensities after the transfer were calculated to be 6x stronger with the Trans-Blot Turbo system compared to the iBlot system 50% decrease in CV — CVs across a single blot were 50% lower with the Trans-Blot Turbo system than with the iBlot system

Intra-blot CV Trans-Blot Turbo system 9% iBlot system 17% Trans-Blot Turbo system

iBlot

Reproducibility across blot. Bio-Rad’s SDS-PAGE Broad Range standards were separated on Criterion 4 –20% gels, and transferred with the Trans-Blot Turbo and Invitrogen’s iBlot systems, both using manufacturers’ recommended 7 min protocol. The nitrocellulose membranes were subsequently stained with SYPRO Ruby and imaged on a VersaDoc™ 4000 MP system.

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2x protein transfer — Quantitation performed on equivalent 2-D gels transferred with the Trans-Blot Turbo and the iBlot systems demonstrated twice the number of proteins transferred and detected with the Trans-Blot Turbo system

2-D Spot Quantitation Trans-Blot Turbo system 1066 iBlot system 555 Trans-Blot Turbo system

iBlot

Higher transfer efficiency using the Trans-Blot Turbo system. Rat liver extract was focused on Bio-Rad’s ReadyStrip™ 11 cm pH 5 – 8 IPG strips and separated on AnykD™ Criterion™ TGX™ gel. Duplicate gels were transferred with the Trans-Blot Turbo and iBlot systems, both using manufacturers’ recommended 7 min protocol. The nitrocellulose membranes were subsequently stained with SYPRO Ruby and imaged on a VersaDoc 4000 MP system.

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www.transblotturbo.com

Ordering Information Catalog #

Description

170-4155 170-4156 170-4157 170-4158 170-4159 170-4151 170-4152

Trans-Blot Turbo Starter System Trans-Blot Turbo Transfer Pack, Mini, PVDF, pkg of 10 Trans-Blot Turbo Transfer Pack, Midi, PVDF, pkg of 10 Trans-Blot Turbo Transfer Pack, Mini, Nitrocellulose, pkg of 10 Trans-Blot Turbo Transfer Pack, Midi, Nitrocellulose, pkg of 10 Trans-Blot Turbo Cassette, single Trans-Blot Turbo Base, no cassettes

Bio-Rad Laboratories, Inc. is licensed by Invitrogen Corporation to sell SYPRO products for research use only under U.S. Patent Number 5,616,502. iBlot is a trademark of Life Technologies. Precision Plus Protein standards are sold under license from Life Technologies Corporation, Carlsbad, CA for use only by the buyer of the product. The buyer is not authorized to sell or resell this product or its components.

Bio-Rad Laboratories, Inc. Web site www.bio-rad.com USA 800 424 6723 Australia 61 2 9914 2800 Austria 01 877 89 01 Belgium 09 385 55 11 Brazil 55 31 3689 6600 Canada 905 364 3435 China 86 20 8732 2339 Czech Republic 420 241 430 532 Denmark 44 52 10 00 Finland 09 804 22 00 France 01 47 95 69 65 Germany 089 31 884 0 Greece 30 210 777 4396 Hong Kong 852 2789 3300 Hungary 36 1 459 6100 India 91 124 4029300 Israel 03 963 6050 Italy 39 02 216091 Japan 03 6361 7000 Korea 82 2 3473 4460 Mexico 52 555 488 7670 The Netherlands 0318 540666 New Zealand 0508 805 500 Norway 23 38 41 30 Poland 48 22 331 99 99 Portugal 351 21 472 7700 Russia 7 495 721 14 04 Singapore 65 6415 3188 South Africa 27 861 246 723 Spain 34 91 590 5200 Sweden 08 555 12700 Switzerland 061 717 95 55 Taiwan 886 2 2578 7189 United Kingdom 020 8328 2000

Life Science Group

Bulletin 6039 Rev A

US/EG

10-1594

0311

Sig 1109


Real-Time PCR: CFX96 Touch System

CFX96 Touch™ Real-Time PCR Detection System Advancing qPCR Together


Real-Time PCR: CFX Automation System

CFX Automation System

The CFX automation system works with the CFX96 Touch™ and CFX384 Touch™ real-time PCR detection systems to enable walk-away, high-throughput operation in a simplified format that does not compromise precision. The plug-and-go benchtop plate handler has the capacity to stack and load up to 20 PCR plates at a time. This system facilitates the automation of workflows, generation of large volumes of data, and rapid data analysis. The system is ideally suited to meet the high-throughput requirements of today’s drug discovery workflows, letting you process up to 7,680 samples in a single run of twenty 384-well plates on the CFX384 Touch system. The CFX automation system makes it easy to: ■■

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Maximize laboratory throughput by integrating 1 CFX96 Touch or CFX384 Touch real-time PCR detection system with hands-free running of up to 20 plates Improve experimental workflow and automation tasks to ensure maximum productivity Track samples using the integrated bar code reader Navigate, set up, and execute multiple PCR experiments using intuitive CFX automation control software

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Define PCR protocols for an entire plate stack at once

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Receive email notification with an attached data file or report upon completion of a single run


Simple Operation and Intuitive Software CFX automation control software manages the configuration and operation of the CFX automation system with the CFX96 Touch or CFX384 Touch real-time PCR detection system, ensuring maximum productivity. The software includes Quick Start and Advanced Run Setup options to program your runs. To get started using the Quick Start option: 1. Select a protocol from the pre-existing list.

Ordering Information Catalog #

Description

184-5072

CFX Automation System, includes robotic plate handler, base tray, bar code scanner, CFX automation control software CD CFX96 Touch Real-Time PCR Detection System, includes C1000 Touch™ thermal cycler chassis, CFX96™ optical reaction module, CFX Manager™ software, license for qbase PLUS software, communication cable, reagents, consumables CFX384 Touch Real-Time PCR Detection System, includes C1000 Touch thermal cycler chassis, CFX384™ optical reaction module, CFX Manager software, license for qbase PLUS software, communication cable, reagents, consumables Hard-Shell® Thin-Wall 384-Well Skirted PCR Plates, clear shell, white well, bar-coded, 50 Hard-Shell Thin-Wall 96-Well Skirted PCR Plates, white shell, clear well, bar-coded, 50

185-5196

185-5484

2. Select a suitable plate setup. 3. Select the number of plates. 4. Click Start to initiate the automation run.

HSP-3905 HSP-9901

Notice regarding Bio-Rad thermal cyclers and real-time systems: Purchase of this instrument conveys a limited non-transferable immunity from suit for the purchaser’s own internal research and development and for use in human in vitro diagnostics and all other applied fields under one or more of U.S. Patent Numbers 5,656,493; 5,333,675; 5,475,610 (Claims 1, 44, 158, 160–163, and 167 only); and 6,703,236 (Claims 1–7 only), or corresponding claims in their non-U.S. counterparts, owned by Applera Corporation. No right is conveyed expressly, by implication or by estoppel under any other patent claim, such as claims to apparatus, reagents, kits, or methods such as 5' nuclease methods. Further information on purchasing licenses may be obtained by contacting the Director of Licensing, Applied Biosystems, 850 Lincoln Centre Drive, Foster City, California 94404, USA. Quick Start window.

Use the Advanced Run Setup option to manage different protocols and plate setups within a batch run.

Bio-Rad’s real-time thermal cyclers are licensed real-time thermal cyclers under Applera’s United States Patent Number 6,814,934 B1 for use in research, human in vitro diagnostics, and all other fields except veterinary diagnostics. Bio-Rad’s real-time thermal cyclers are covered by one or more of the following U.S. patents or their foreign counterparts owned by Eppendorf AG: U.S. Patent Numbers 6,767,512 and 7,074,367. Hard-Shell plates are covered by one or more of the following U.S. patents or their foreign counterparts owned by Eppendorf AG: U.S. Patent Numbers 7,347,977; 6,340,589; and 6,528,302.

Advanced Run Setup window.

Bio-Rad Laboratories, Inc. Web site www.bio-rad.com USA 800 424 6723 Australia 61 2 9914 2800 Austria 01 877 89 01 Belgium 09 385 55 11 Brazil 55 31 3689 6600 Canada 905 364 3435 China 86 21 6169 8500 Czech Republic 420 241 430 532 Denmark 44 52 10 00 Finland 09 804 22 00 France 01 47 95 69 65 Germany 089 31 884 0 Greece 30 210 777 4396 Hong Kong 852 2789 3300 Hungary 36 1 459 6100 India 91 124 4029300 Israel 03 963 6050 Italy 39 02 216091 Japan 03 6361 7000 Korea 82 2 3473 4460 Malaysia 60 3 2117 5260 Mexico 52 555 488 7670 The Netherlands 0318 540666 New Zealand 64 9 415 2280 Norway 23 38 41 30 Poland 48 22 331 99 99 Portugal 351 21 472 7700 Russia 7 495 721 14 04 Singapore 65 6415 3170 South Africa 27 861 246 723 Spain 34 91 590 5200 Sweden 08 555 12700 Switzerland 061 717 95 55 Taiwan 886 2 2578 7189 Thailand 66 2 6518311 United Kingdom 020 8328 2000

Life Science Group

Bulletin 5879 Rev B

US/EG

11-0133

0311

Sig 0211


Thermal Cyclers: C1000 Touch™ Thermal Cycler

Advancing PCR Together PCR Faster n See accurate, reliable results with shorter run times and superior thermal performance n

Save time by optimizing annealing temperature in a single run using a temperature gradient

PCR Easier Easily edit and run protocols using the large color touch screen

n

n

n

n

Choose the best setup for your needs from 6 interchangeable reaction modules, including 2 optical modules for real-time PCR Increase throughput as your research grows — control of 2–32 instruments gives you the flexibility you need Use a wide range of reaction vessels with the fully adjustable heated lid

PCR Smarter Quickly create protocols with 2 easy programming options: graphical and automatic with the protocol autowriter

n

n

Get reliable results for years with a patented* O-ring seal that protects thermal electric modules

For more information, visit us on the Web at www.bio-rad.com/1000-series. * U.S. patent 7,051,536.


Amplification: Thermal Cyclers

1000-Series Thermal Cyclers Advancing qPCR Together


Amplification © 2011 Bio-Rad Laboratories, Inc. All rights reserved.

CFX Manager™ Software Version 2.1


Electrophoresis and Blotting

Protein Blotting Guide

BEGIN


Protein Blotting Guide

Theory and Products

MORE INFORMATION

Part 1 Theory and Products

5

Chapter 1 Overview of Protein Blotting

5

Transfer

6

Detection

6

General Considerations and Workflow

6

Chapter 2 Methods and Instrumentation

9

Protein Blotting Methods Electrophoretic Transfer Tank Blotting Semi-Dry Blotting

Total Protein Detection

10 10 10 11

Microfiltration (Dot Blotting)

Blotting Systems and Power Supplies Tank Blotting Cells Mini Trans-Blot® Cell and Criterion™ Blotter Trans-Blot® Cell Trans-Blot® Plus Cell

Semi-Dry Blotting Cells Trans-Blot® SD Semi-Dry Cell Trans-Blot® Turbo™ System

Microfiltration Apparatus Bio-Dot® and Bio-Dot® SF Apparatus

TABLE OF CONTENTS

Power Supplies for Electrophoretic Transfers PowerPac™ HC Power Supply PowerPac™ Universal Power Supply

Chapter 3 Membranes and Transfer Buffers Membranes and Blotting Papers Nitrocellulose and Supported Nitrocellulose Polyvinylidene Difluoride (PVDF) Immun-Blot®

and Immun-Blot LF PVDF for Western Blotting Sequi-Blot ™ PVDF for Protein Sequencing

Blotting Filter Papers Membrane/Filter Paper Sandwiches

Transfer Buffers Towbin and Bjerrum Schafer-Nielsen Buffers (Tris/Glycine Buffers) CAPS Buffer Discontinuous Tris-CAPS Buffer System (for Semi-Dry Transfer) Dunn Carbonate Buffer Other Buffers

12 12 12 12 13 13 14 14 14 14 15 15 15 17 18 18 18 18 18 19 19 19 20 20 20 21 21

Chapter 4 Transfer Conditions

23

General Workflow – Electrophoretic Transfer

24

Power Conditions

24 24 24 24 High-Intensity Field Transfers 24 Standard Field Transfers 26 Selecting Power Supply Settings 26 Transfers Under Constant Voltage 26 Transfers Under Constant Current 26 Transfers Under Constant Power 26 General Guidelines for Transfer Buffers and Transfer Conditions 26 Useful Equations Joule Heating and Other Factors Affecting Transfer Relationship Between Power Settings and Transfer Times

2

Chapter 5 Detection and Imaging Anionic Dyes Fluorescent Stains Stain-Free Technology Collodial Gold

Immunodetection Immunodetection Workflow Blocking Antibody Incubations Washes

Antibody Selection and Dilution Primary Antibodies Species-Specific Secondary Antibodies Antibody-Specific Ligands

Detection Methods Colorimetric Detection Premixed and Individual Colorimetric Substrates Immun-Blot Assay Kits Immun-Blot Amplified AP Kit Opti-4CN™ and Amplified Opti-4CN Substrate and Detection Kits Chemiluminescence Detection Immun-Star ™ Chemiluminescence Kits Fluorescence Detection Other Detection Methods Bioluminescence Chemifluorescence Autoradiography Immunogold Labeling Stripping and Reprobing

Imaging — Analysis and Documentation Luminescence Detection Digital Imaging for Fluorescence, Chemifluorescence, and Colorimetric Detection Autoradiography Analysis Software

Part 2 Methods Protocols Electrophoretic Transfers Reagent and Materials Preparation Tank Blotting Procedure Prepare the Gel and Membrane Sandwich Assemble the Tank and Program the Power Supply Semi-Dry Blotting Procedure Trans-Blot Turbo Blotting Procedure

Microfiltration Blot Stripping and Reprobing Total Protein Detection SYPRO Ruby Stain Ponceau S Stain Colloidal Gold Total Protein Stain

Immunodetection Notes for Multiplex Detection Notes for Chemiluminescence Detection Notes for Fluorescence Detection Notes for Protein G-HRP Detection Notes for Amplified Opti-4CN Detection Notes for Amplified AP Detection

29 31 31 31 32 32 32 33 33 33 33 34 34 34 34 35 36 38 38 38 38 40 40 41 41 42 42 42 42

Transfer Buffer Formulations Towbin Buffer Towbin Buffer with SDS Bjerrum Schafer-Nielsen Buffer Bjerrum Schafer-Nielsen Buffer with SDS CAPS Buffer Dunn Carbonate Buffer 0.7% Acetic Acid

TIPS

Detection Buffer Formulations General Detection Buffers Total Protein Staining Buffers and Solutions Substrate Buffers and Solutions Stripping Buffer

Part 3 Troubleshooting

58 58 58 58 58 58 58 58 58 58 59 60 60 63

Transfer

64 64 65

Electrophoretic Transfer Microfiltration

Detection

66 66 68 68

Immunodetection Multiscreen Apparatus Total Protein Detection

Appendix

70

Protein Standards for Blotting Unstained Standards for Protein Blotting Unstained SDS-PAGE Standards Precision Plus Protein™ Unstained Standards

43 43

Prestained Standards for Western Blotting

44 44 44

Precision Plus Protein™ WesternC™ Standards

Precision Plus Protein Prestained Standards Kaleidoscope™ Standards Prestained SDS-PAGE Standards

70 71 71 71 72 72 72 72 73

Glossary

74

47

References and Related Reading

78

48 48 48 49 49 50 51 52 53 54 55 55 55 55 56 56 57 57 57 57 57

Ordering Information

80

Links:

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Protein Blotting Guide

Theory and Products

MORE INFORMATION

PART 1

Theory and Products CHAPTER 1 TABLE OF CONTENTS

Overview of Protein Blotting Protein blotting, the transfer of proteins to solid-phase membrane supports, is a powerful and popular technique for the visualization and identification of proteins. When bound to membranes, proteins are readily accessible for immunological or biochemical analyses, quantitative staining, or demonstration of proteinprotein or protein-ligand interactions. This chapter provides an overview of the methods and workflow of protein blotting, which involves two phases: transfer and detection. 4

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Designed.