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GE Healthcare

Discovery Matters

Innovations for the life scientist from GE Healthcare Issue 9, February 2009

GE & Science Prize for Young Life Scientists 2008 Concentrating protein for X-ray crystallography Label-free characterization of anti-drug antibodies Isolating nucleic acids and protein from undivided samples

imagination at work


GE Healthcare

Discovery Matters

Innovations for the life scientist from GE Healthcare Issue 9, February 2009

contents What’s New? Details of our latest products and services

3–8

GE & Science Prize for Young Life Scientists 2008 Concentrating protein for X-ray crystallography Label-free characterization of anti-drug antibodies Isolating nucleic acids and protein from undivided samples

Technical Tips A simple, two-step purification method designed for purification of histidine-tagged membrane proteins

9

imagination at work

Issue 9, February 2009

Technology Central Convenient and efficient methods for cell lysis and protein extraction

Editor Alexander Razdan, PhD Associate Editors John Osborn, BSc Oki Dzivenu, DPhil Jim Malone, MSc Christina Ninalga, PhD Production Pär Jansson Discovery Matters is published by GE Healthcare. The goal of Discovery Matters is to provide you with information that will help you achieve your research objectives. We want to continue developing Discovery Matters into a publication you value, and appreciate your input. Please send your articles for submission in upcoming issues, plus any comments or questions to: alex.razdan@ge.com

10–11

Innovations Forum Protein sample preparation Convenient and rapid concentration of protein solutions for X-ray crystallography using Vivaspin sample concentrators

12–13

Label-free interaction analysis Reliable detection and characterization of anti-drug antibodies using Biacore T100 Immunogenicity Package

14–15

Nucleic acid purification Comparative analysis of illustra triplePrep Kit for the isolation of genomic DNA, total RNA, and total denatured protein from a single undivided sample

16-17

GE & Science Prize for Young Scientists 2008 Understanding a minimal DNA-segregating machine Reconstitution of a plasmid spindle shows how three components together accomplish the task of DNA segregation

18–19

To receive Discovery Matters, subscribe online at: www.gelifesciences.com The content of this issue and previous issues of Discovery Matters can also be viewed online at www.gelifesciences.com/ discoverymatters

Have you seen the Products for Life Sciences 2008/2009 catalog? The new catalog is a comprehensive guide to the majority of products for life science research from GE Healthcare. Order your copy online at: www.gelifesciences.com/catalog

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What’s New?

Mammalian Protein Extraction Buffer and Yeast Protein Extraction Buffer Kit Mammalian Protein Extraction Buffer and Yeast Protein Extraction Buffer Kit offer a convenient method to extract total soluble protein from mammalian cultured cells and yeast cells, respectively. In the past, physical methods of cell disruption were commonly used which were laborious, vigorous, and often required expensive equipment. The Mammalian Protein Extraction Buffer and Yeast Extraction Buffer Kit provide mild, detergent-based cell lysis, eliminating the need for mechanical cell lysis, and delivering high quality protein lysate compatible with most downstream applications. Key benefits include: >> High protein recovery: proprietary formulations give efficient protein extraction >> Maintained biological activity: mild, non-denaturing buffer compositions

>> Convenience: ready-to-use buffers save time by eliminating the need for homemade buffers. The Yeast Protein Extraction Buffer Kit allows for efficient extraction without having to use glass beads

Yeast Protein Extraction Buffer Kit

The 2-D Protein Extraction Buffer Trial Kit contains each of the six extraction buffers, allowing you to screen for the most appropriate extraction buffer for your sample. The extraction buffers can also be used prior to 1-D electrophoresis, or for rehydrating IPG strips prior to 2-D electrophoresis. Key benefits include:

>> High protein yield: mix of chaotropic agents and detergents results in efficient protein extraction >> High reproducibility: high quality reagents increase consistency and reliability

Ordering information

Ordering information Mammalian Protein Extraction Buffer

Efficient cell lysis and protein extraction are key steps for achieving high quality results in downstream applications such as 2-D gel analysis. The 2-D Protein Extraction Buffers offer a convenient way to prepare high quality protein lysates. Six protein extraction buffers are available, all of which are modifications of well-proven protein extraction buffers designed to produce high spot resolution for 2-D gel analysis. The buffers are provided in a dry powder formulation, eliminating carbamylation, a reaction that can occur in solution with urea to alter protein charge and produce spot artifacts on 2-D spot maps. With 2-D Protein Extraction Buffers, however, buffer can be freshly made by weighing out the necessary amount and resuspending it in the included DILUENT.

>> Simplified preparation: save time using ready to mix buffers

>> High reproducibility: high quality reagents produce consistent results

Product

2-D Protein Extraction Buffers

Code number

Product

Code number

28-9412-79

2-D Protein Extraction Buffer-I

28-9435-23

28-9440-45

2-D Protein Extraction Buffer-II

28-9435-24

2-D Protein Extraction Buffer-III

28-9435-25

2-D Protein Extraction Buffer-IV

28-9435-26

2-D Protein Extraction Buffer-V

28-9435-27

2-D Protein Extraction Buffer-VI

28-9435-28

2-D Protein Extraction Buffer Trial Kit

28-9435-22

For more information, visit www.gelifesciences.com/sampleprep

For more information, visit www.gelifesciences.com/sampleprep

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What’s New?

DeCyder 2D Differential Analysis Software v7.0

illustra triplePrep Kit

>> Isolate genomic DNA (gDNA), total RNA, and total denatured proteins from undivided tissue and cell samples in less than 1 h >> Directly correlate DNA, RNA, and protein data from the same sample >> Obtain high yield gDNA, RNA, and protein from precious limited samples >> Flexible workflow allows easy isolation of any two or all three analytes >> Color-coded caps and bottles with matching protocol steps minimize error illustra™ triplePrep Kit is designed for rapid simultaneous extraction and purification of high quality gDNA, total RNA, and total denatured proteins from animal tissues and mammalian cells. The streamlined workflow reduces the overall number of steps, resulting in up to 70% time saving compared to preparing each analyte individually. The highly purified gDNA, RNA, and protein obtained with the illustra triplePrep Kit are suitable for downstream genomic and proteomic applications such as PCR, restriction digestion, sequencing, array CGH, RT-qPCR, gene expression microarray, SDS-PAGE, Western blotting, 2-D DIGE, and LCMS. The optimized buffer, columns, and protocol ensure high recovery of gDNA, RNA, and proteins enabling the use of precious limited samples such as biopsies, archived tissues, and tumors.

illustra triplePrep Kit, 50 preps

In v7.0, the Extended Data Analysis (EDA) Software is included in the standard software package, allowing the user to further characterize biological samples by performing advanced statistical analyses. EDA also enables the user to link data sets, in order to increase the sample size and more easily differentiate between real and experimental variation. DeCyder 2D software is compatible with Ettan™ DIGE Imager and Typhoon™ variable mode imagers. What’s new in v7.0: >> Warping: improves matching accuracy >> Image Editor: easily extract raw images for analysis or high resolution images for publishing >> “Save as” function: create alternative statistical analyses without affecting previous analyses >> Improved image functionality: facilitates spot matching and increases user friendliness

Ordering information

Ordering information Product

DeCyder™ 2D Differential Analysis Software v7.0 performs full analysis of differences in protein expression from 2-D fluorescence difference gel electrophoresis (2-D DIGE) experiments. 2-D DIGE is a powerful tool for detection and quantitation of real biological differences in protein abundance between different samples. DeCyder 2D software increases throughput by reducing handson time from days to minutes, with minimal user-to-user variation. The software provides accurate, reliable data and performs analyses with superior statistical confidence. Tests show that DeCyder 2D software identifies all differences with negligible statistical error.

Code number 28-9425-44

For more information on our range of protein and nucleic acid sample preparation products, visit www.gelifesciences.com/illustra

Product

Code number

DeCyder 2D Oracle 10gR2, 5-user license

28-9435-88

DeCyder 2D 7.0, additional single-user license

28-9442-77

DeCyder 2D 7.0, single-user trial license

28-9442-79

DeCyder 2D 7.0, single-user license (upgrade from 6.5 2D)

28-9442-80

DeCyder 2D 7.0, single-user license upgrade from 6.5

28-9442-81

Image Master 2D Platinum 6.0 DIGE upgrade to 7.0 DIGE, 1 license

28-9380-80

For more information, visit www.gelifesciences.com/decyder

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What’s New?

ImageMaster 2D Platinum v7.0

ImageQuant TL SecurITy Software Image added to Secure Folder

Working copy created

Analysis performed in 1D gel analysis module

Data stored in Secure Folder (version number obtained)

No

Analysis complete? Yes

Results reviewed

No

Results acceptable? Yes

Data approved

ImageMaster™ 2D Platinum v7.0 is a flexible software package for visualization and analysis of 2-D gel data, including basic 2-D DIGE (2-D fluorescence difference gel electrophoresis) and classical 2-D methodologies. 2-D DIGE is a system that offers significant advantages over classical 2-D electrophoresis, and ImageMaster 2D Platinum software uses a patented co-detection algorithm to analyze 2-D DIGE results. With interactive analysis modes and an improved user interface, v7.0 offers significant new features for greater convenience and speed, simplifying even the most complex analysis. Key features of ImageMaster 2D Platinum v7.0 include: >> Well-established methods that can be applied to any 2-D experiment

ImageQuant™ TL SecurITy is a supplementary software package designed to enhance data security in the ImageQuant TL software. ImageQuant TL SecurITy software allows for greater control and traceability of 1-D gel electrophoresis data analysis. ImageQuant TL SecurITy offers added security features provided by the Version Control Tool and Admin Tool. The Admin Tool is used to set up user security and restrict user access to image files, while the Version Control Tool manages multiple revisions of analyzed 1-D gel images. ImageQuant TL SecurITy is compatible with all imaging systems from GE Healthcare.

>> Effective spot detection and matching

ImageQuant TL SecurITy software includes:

>> User-friendly and flexible interface

>> User name and password protection to restrict access to authorized individuals

>> Seamless integration into laboratory workflow >> Versatile visualization tools that simplify image handling

Ordering information Product

Code number

ImageMaster 2D Platinum 7.0 software package

28-9408-30

Image Master 2D Platinum 7.0, 1 license

28-9380-91

Image Master 2D Platinum 7.0 DIGE, 1 license

28-9380-55

Image Master 2D Platinum 7.0 upgrade to DIGE, 1 license

28-9398-10

Image Master 2D Platinum 6.0 upgrade to 7.0, 1 license

28-9399-70

Image Master 2D Platinum 6.0 DIGE upgrade to 7.0 DIGE, 1 license

28-9380-80

>> Approval of experiments with electronic signatures >> Version control audit trail ensures traceability of multiple versions of gel images >> Experiment audit reports provide documentation and simplify tracking of image analysis data

Ordering information Product

Code number

ImageQuant TL 7.0 and ImageQuant TL SecurITy 8.0 Software Package (with Getting Started Guide)

28-9380-94

ImageQuant TL 7.0 and ImageQuant TL SecurITy 8.0, single user license

28-9236-62

ImageQuant TL SecurITy 8.0, single user license upgrade

28-9398-15

For more information and a 14 day free trial of ImageMaster 2D Platinum, visit www.gelifesciences.com/imp For more information, visit www.gelifesciences.com/iqtl

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What’s New?

KappaSelect

Heavy chain Light chain

Heavy chain Light chain VL

FabVH

VL

Fab

VH

VH

V CL –S–S– CH H

CVHL –S–S

VL

Biacore T100 Immunogenicity Package

CL

CH –S–S –S–S– CL

CL –S–S– CH

–S–S–

Fc

–S–S– –S–S–

IgG

Fc IgG CH

VH

CH

VH

CL

Fab

CL

Fab

Binding site for KappaSelect ligand

VH Binding site for KappaSelect ligand CH

CH

VH

VH

CL

VL

VL CL

VL

VL

VH

VL

CL

VL –S–S–

CH

CH

CL

F(ab)2 = Papain digestion

–S–S–

F(ab)2

= Pepsin digestion —S—S—

= Disulfide bridge = Carbohydrate

= Papain digestion = Pepsin digestion —S—S—

= Disulfide bridge

Antibody structure and binding site for KappaSelect ligand to = Carbohydrate Fab fragment.

KappaSelect is an affinity chromatography medium designed as a Custom Designed Media for the purification of Fab (kappa) antibody fragments. The ligand binds to the constant region of the kappa light chain enabling an efficient capture step for antibody fragments such as Fabs with high purity and yield.

Biacore™ T100 Immunogenicity Package provides tools for the reliable detection and characterization of anti-drug antibodies (ADAs) during preclinical and clinical development. In addition to detecting low affinity antibodies often associated with an early immune response, the package addresses drug interference by enabling measurement of ADAs in the presence of excess amounts of drug. New software tools are also provided for comprehensive characterization of ADAs. Biacore T100 Immunogenicity Package is sold as an optional module for Biacore T100. Biacore T100 Immunogenicity Package allows for: >> Reliable detection of ADAs: Detect ADAs in the presence of drug and minimize false negatives by detecting even low affinity ADAs

KappaSelect affinity chromatography medium provides:

>> Comprehensive characterization of immune response: Easily determine antibody class or subclass and binding stability

>> Efficient, industrial-scale capture of Fabs by affinity chromatography

>> Reduced assay development time: Assay guidelines, recommendations, and support decrease startup time

>> High binding capacity for Fabs >> Rigid agarose base matrix for high flow rates, allowing processing of large sample volumes for increased throughput

Ordering information

>> Non-mammalian derived product, which reduces regulatory concerns in the production of Fabs for clinical applications

Biacore T100 Immunogenicity Package Includes: Biacore T100 Immunogenicity Software, License Agreement with product key, Biacore T100 Immunogenicity Handbook – including assay guidelines and recommendations

>> Low ligand leakage, which ensures increased Fab purity and productivity

Ordering information Product

Code number 28-9423-71

For more information, visit www.gelifesciences.com/biacore Code number

KappaSelect, 25 ml

17-5458-01

KappaSelect, 200 ml

17-5458-02

KappaSelect, 1 l*

17-5458-03

* Larger pack sizes are available, please contact your local GE Healthcare representative

For more information, visit www.gelifesciences.com/cdm

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Product


What’s New?

Updated Recombinant Protein Purification Handbook

Extended range of HiPrep Sephacryl HR gel filtration columns

The Recombinant Protein Purification Handbook: Principles and Methods has been revised. Main changes include addition of two chapters, creation of additional selection guides, and expansion of the desalting chapter to describe new, highthroughput products plus Vivaspin™ sample concentrators. The first new chapter describes tagging proteins with maltose binding protein and affinity purification using Dextrin Sepharose™ High Performance. The second new chapter describes use of the Strep-tag™ II peptide tag and affinity purification using StrepTactin Sepharose High Performance. Both of these chapters plus the expanded desalting chapter include selection guides and application examples. Updates were also made in other chapters to include new products and/or applications.

Ordering information Product Recombinant Protein Purification Handbook: Principles and Methods

Code number 18-1142-75

Visit www.gelifesciences.com/handbooks to download a copy

HiPrep™ Sephacryl™ S-400 HR and S-500 HR columns are the latest additions to the growing range of columns for chromatographic purification of molecules by gel filtration (size-exclusion chromatography). HiPrep Sephacryl HR columns enable a fast and convenient approach to the purification of large proteins, polysaccharides, and macromolecules with extended structures. The expanded range of columns allows purification of molecules with molecular weights as small as 1 × 103 to macromolecules with molecular weights as large as 2 × 107. Two sizes of each are new column are available: 16/60 (column volume 120 ml) for sample volumes of up to 5 ml and 26/60 (column volume 320 ml) for sample volumes of up to 13 ml. HiPrep Sephacryl HR columns are prepacked with BioProcess™ validated chromatography media, which are tailored for use in both laboratory and industrial-scale applications. In addition, HiPrep Sephacryl HR columns provide: >> Easy connection to chromatography systems such as the range of ÄKTAdesign™ systems >> Reliable and reproducible purifications >> Convenient scale-up

Ordering information Product

Code number

HiPrep 16/60 Sephacryl S-400 HR

28-9356-04

HiPrep 26/60 Sephacryl S-400 HR

28-9356-05

HiPrep 16/60 Sephacryl S-500 HR

28-9356-06

HiPrep 26/60 Sephacryl S-500 HR

28-9356-07

For more information, visit www.gelifesciences.com/protein-purification

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GE & Science Prize 2008 GE & Science Prize for Young Life Scientists 2008; winners pictured at the Karolinska Institute in Stockholm with Nobel Laureates in Medicine, Françoise BarréSinoussi and Luc Montagnier. Back row from left to right: Sabrina Büttner, Ethan Clark Garner (Grand Prize winner), and Sarel Fleishman. Front row from left to right: Luc Montagnier, Françoise Barré-Sinoussi, Kaori Yamada.

GE & Science Prize for Young Life Scientists 2008 Established in 1995, the GE & Science Prize for Young Life Scientists seeks to bring science to life by recognizing outstanding PhDs from around the world, and rewarding their research in the field of molecular biology. Both Science/AAAS and GE Healthcare believe that support of promising scientists at the beginning of their careers is critical for continued scientific progress. This year there were over 100 high-caliber entrants, which made the judging process more challenging than ever. Entrants submit a 1000 word synopsis of their thesis to the journal Science for judging by an independent, scientific panel of judges. Each year, one Grand Prize winner is selected, winning $25 000 in prize money and up to four regional prizes are also awarded, each receiving $5000. The Grand Prize winner has his/her essay published in Science and the regional winners in the online version of Science. The winners attended the awards ceremony held at Stockholm’s Grand Hotel. In addition, the prize-winners met the Nobel Laureates in Medicine, Françoise Barré-Sinousi, Luc Montagnier, and Harald zur Hausen at the Nobel Q&A session and lunch hosted by Karolinska Institute in Stockholm. During the two-day event, the award winners also visited the facilities at GE Healthcare and Uppsala University.

2008 Grand Prize winner Ethan Clark Garner, the author of the prize-winning essay, “Understanding a DNA-segregating machine”, was born in Richland, Washington. He received his BSc in biochemistry from Washington State University, where he worked with Keith Dunker developing tools to predict disordered regions within proteins. He conducted his graduate work at the University of California, San Francisco, where he studied the kinetics and regulation of prokaryotic polymers with Dyche Mullins. Ethan has since moved to Boston, where he will be working with Tim Mitchinson, Xiaowei Zhuang, and Alice Ting on elucidating the process of prokaryotic DNA segregation.

Regional Winners North America: Xu Tan for his essay “Plant Hormone Auxin Functions as Novel Molecular Glue”. Dr. Tan spent his first 18 years in Changsha, China. He became hooked on biology in high school and won a national first prize in the biology Olympiad. After earning his BSc from the University of Science and Technology of China in Hefei, he pursued graduate studies at the University of Washington, Seattle. Under the advice of Ning Zheng, Dr. Tan did his thesis research on the structural biology of

8

ubiquitin ligases. Looking forward to expanding his research horizons, he is starting a postdoctoral position with Steve Elledge at Brigham and Women’s Hospital, Harvard Medical School. Europe: Sabrina Büttner for her essay “Endonuclease G Regulates Cellular Fate”. Dr. Büttner was born in Mutlangen, Germany. She studied biochemistry at the Eberhardt-Karls University, Tübingen, Germany, and received her diploma with honors in 2004. During her PhD studies, conducted under the guidance of Frank Madeo at the Institute for Molecular Biosciences, University of Graz, Austria, she investigated yeast programmed cell death in the context of aging and oxidative stress, identifying molecular mechanisms of apoptosis in S. cerevisiae. After defending her doctoral thesis in 2007, Dr. Büttner continued her research in the Madeo lab as a postdoctoral fellow, focusing on the further establishment of yeast as a model for neurodegenerative diseases. Japan: Kaori Yamada for her essay “Moving PIP3 Regulates Cell Polarity.” Dr. Yamada grew up in the town of Kinokawa, Japan. She received a BSc from the University of Tokyo. A strong interest in life science led her to remain there as a graduate student in Yasuhisa Fukui’s laboratory. During her postgraduate project, she spent time at the laboratory of Athar H. Chishti, a collaborator at the University of Illinois, Chicago. There, Dr. Yamada elucidated how kinesin transports the lipid messenger PIP3 in neurons. She completed her PhD in January 2007 and is currently a postdoctoral fellow at the University of Illinois, Chicago. All other countries: Sarel Fleishman for his essay “Modeling at the Gates of the Cell.” Dr. Fleishman received an MSc in biochemistry (summa cum laude) and a PhD (with distinction) from Tel-Aviv University, Israel, where he studied in the group of Nir Ben-Tal. During his graduate studies, he investigated the structure, function, and evolution of membrane proteins associated with hereditary hearing loss and neurodegenerative diseases, cancer, and bacterial drug resistance. He is currently a Human Frontier Research Postdoctoral Fellow working on computational design of protein-based inhibitors toward pathogenic molecules in David Baker’s laboratory at the University of Washington. The Grand Prize winning essay, published on page 18 of this issue of Discovery Matters, is reprinted with permission from AAAS. For the full text of essays by the regional winners and to apply for this year’s awards, visit www.sciencemag.org/feature/data/prizes/ge


Starting with cells expressing a histidine-tagged putative transferase membrane protein, lysis and membrane solubilization was performed by chemical and freeze-thaw methods in a lysis buffer containing 1% FC12 detergent. The unclarified lysate was then applied directly to HisTrap FF crude 1 ml connected to ÄKTAexplorer™ chromatography system for affinity purification of the tagged protein (Fig 1A). Elution was performed with buffers containing the detergents, FC12, TCEP, and DDM. The eluate from HisTrap FF crude 1 ml step was applied to HiLoad 16/60 Superdex 200 pg. In this step, remaining contaminants as well as imidazole used for elution from the first chromatography step were removed using a buffer containing TCEP and DDM. A preliminary size estimation of the target protein achieved at the same time revealed that gel filtration effectively removed most contaminants and showed that no protein aggregates were present in the sample (Fig 1B). To determine target protein purity, pooled fractions were analyzed by SDS-PAGE (Fig 1C). Purification of the membrane protein as described briefly here ensures a completely pure protein, free from contaminants that would otherwise interfere with further analysis of the protein by, for example, X-ray crystallography. This basic method can be modified to allow purification of other membrane proteins by adjusting detergent composition, detergent concentration, or pH.

Column: Equilibration: Sample: Binding buffer:

HisTrap FF crude 1 ml Binding buffer 5 ml unclarified E. coli cell lysate with 1% FC12 (detergent) 20 mM sodium phosphate, 500 mM sodium chloride, 20 mM imidazole, 0.5 mM TCEP, 1% FC12, pH 7.4 20 mM sodium phosphate, 500 mM sodium chloride, 40 mM imidazole, 0.5 mM TCEP, 0.03% dodecylmaltoside (DDM), 1% FC12, pH 7.4 Wash buffer with 500 mM imidazole ÄKTAexplorer

Wash:

Elution buffer: System:

A)

mAU 3500 3000 2500 2000 1500 1000 mAU 500 3500 0 3000 2500

0.0

5.0

10.0

15.0

20.0

25.0

ml

2000 1500 mAU

Column:1000 700 500 Equilibration: 600 Sample: 5000 Elution: 400 System: 0.0

HiLoad 16/10 Superdex 200 pg 20 mM Tris-HCl, 50 mM NaCl, 0.5 mM TCEP, 0.03% DDM, pH 8.0 Pooled fractions selected from affinity purification step Equilibration buffer, see above 5.0 10.0 15.0 20.0 25.0 ml ÄKTAexplorer

300

B)

200 mAU 100 700 0 600 500 0

20

40

60

80

100

ml

20

40

60

80

100

ml

400 300 200

Although this article describes purification using an ÄKTAexplorer chromatography system, a faster, fully automated approach to the purification of tagged membrane proteins can be achieved using ÄKTAxpress™ system.

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The data shown in this Technical Tip were kindly provided by Dr. S. Eshaghi, Karolinska Institute, Stockholm, Sweden.

Product

TECHNICAL TIPS

Fast and efficient purification of histidine-tagged membrane proteins can be performed by a combination of affinity chromatography on HisTrap™ FF crude 1 ml column and gel filtration (size-exclusion chromatography) on HiLoad™ 16/60 Superdex™ 200 pg. This is a fast and generic purification protocol that does not require optimization. The imidazole used for elution in the first step is removed during the gel filtration step together with protein aggregates to give a highly pure product.

Protein sample preparation

A simple, two-step method for purification of histidine-tagged membrane proteins

Mr × 103 97

A detailed description of this method can be downloaded from www.gelifesciences.com, article code number 28-9490-15.

66 45 30 20.1 14.4

Fig 1. (A) Affinity chromatography of a histidine-tagged putative transferase membrane protein on HisTrap FF crude 1 ml column. (B) Gel filtration of fractions from A) removed low molecular weight contaminants including imidazole, and aggregates. (C) SDS-PAGE (Coomassie™ stained gel) of the target protein after the two-step purification.

9


GE Healthcare brings simplicity and versatility to protein extraction from complex samples prior to downstream protein analysis methods such as liquid chromatography, 2-D electrophoresis, and 2-D DIGE. Mammalian Protein Extraction Buffer is designed for efficient extraction of total soluble protein from both adherent and nonadherent mammalian cell cultures. Yeast Protein Extraction Buffer Kit is developed for mild extraction of soluble proteins from yeast cells. The 2-D Protein Extraction Buffers are six different solubilization buffers designed to produce high spot resolution for 2-D gel analysis. The 2-D Protein Extraction Buffer Trial Kit allows you to evaluate each extraction buffer to find the most suitable buffer for your study.

A)

Buffer kits for extraction of mammalian and yeast proteins

B)

Mammalian Protein Extraction Buffer and Yeast Protein Extraction Buffer are based on organic buffering agents, mild nonionic detergents, and a combination of various salts and agents to gently disrupt the cell wall and release soluble proteins. Depending on the application, additional agents such as chelating agents, reducing agents, or protease inhibitors may be added directly to the extraction buffers before use. Both buffers are compatible with most downstream applications including chromatography, electrophoresis, immunoassays, and enzyme assays. The Yeast Protein Extraction Buffer Kit eliminates the need for glass beads, a common method of mechanical lysis for yeast cells. A readyto-use Zymolyase™ preparation and Yeast Suspension Buffer are also provided in the kit. The Yeast Suspension Buffer is used during cell lysis, together with Zymolyase, an enzyme that digests the cell wall layer of yeast. After lysis, Yeast Protein Extraction Buffer is added to the yeast pellet, referred to as spheroplast, to extract biologically active, soluble protein.

10

2500

Yield (µg)

2000 1500 1000 500 0

1000

800

Yield (µg)

technology CENTRAL

Convenient and efficient methods for cell lysis and protein extraction

600

400

200

0

High reproducibility using (A) Mammalian Protein Extraction Buffer and (B) Yeast Protein Extraction Buffer Kit. Total yield for the technical replicates is shown, as well as the average and standard deviation. In the extraction using Mammalian Protein Extraction Buffer, 0.3 ml was used to lyse and extract 10 samples containing 1 × 107 CHO cells. One sample is not shown due to bubbles in the well. In the extraction using Yeast Protein Extraction Buffer Kit, 0.1 ml was used to extract 10 samples containing 50 mg of S. cerevisiae.


2-D Protein Extraction Buffers allow for convenient buffer preparation and efficient extractions that result in high quality 2-D electrophoresis results. All six buffers are compatible with 1-D and 2-D electrophoresis under denaturing conditions and reagents such as protease inhibitor cocktails may be added to the buffers. Most buffers are compatible with CyDye™ DIGE Fluors used in 2-D fluorescence difference gel electrophoresis (2-D DIGE)1. Tissue sample ≤ 100 mg

The 2-D Protein Extraction Buffer Trial Kit is available to help you determine the most suitable extraction buffer for your study. Because some protein spots will be differentially extracted by the buffers, the most appropriate extraction buffer will depend on the nature of your sample and the purpose of your study. 1

2-D Protein Extraction Buffer-I is not recommended when using CyDye DIGE Fluor minimal dyes and Extraction Buffer–III and –IV are not suitable when using CyDye DIGE Fluor Labeling Kit for Scarce Samples.

2-D Protein Extraction Buffer

Homogenization - Sample Grinding Kit

technology CENTRAL

Protein extraction buffers for 2-D electrophoresis

Removal of contaminants - 2-D Clean-up Kit

Quantitation of protein lysate - 2-D Quant Kit

Sample labeling CyDye DIGE Fluor minimal dyes, CyDye DIGE Fluor for Scarce Samples

2-D electrophoresis - Immobiline™ DryStrip gels, SDS-PAGE gels

Image capture - Ettan™ DIGE Imager/Typhoon™

Image analysis software - DeCyder™

Screening for optimal protein extraction using 2-D Protein Extraction Buffers. In this 2-D protein spot map, spots of protein seen in red were extracted to a higher degree with Extraction Buffer-II, while spots of protein seen in green were more highly abundant after extraction with Extraction Buffer-VI.

For more detailed information on products for sample preparation, visit www.gelifesciences.com/sampleprep For more information about 2-D gel electrophoresis, visit www.gelifesciences.com/DIGE

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Protein sample preparation

Innovations Forum

Convenient and rapid concentration of protein solutions for X-ray crystallography using Vivaspin sample concentrators T. Granér and R. Bhikhabhai GE Healthcare, Uppsala, Sweden

Protein concentration is a crucial step prior to crystallization to obtain diffraction-quality crystal for X-ray structure determination. In this study, a two-step purification of histidine-tagged protein upstream of crystallography is described. Vivaspin™ sample concentrators were used to concentrate the sample between the two purification steps, as well as for buffer exchange and sample concentration prior to crystallization.

Introduction In structural genomics, a two-step purification, affinity chromatography followed by gel filtration (size-exclusion chromatography), is often used. Frequently, sample volume must be reduced before the second purification step, gel filtration, where size heterogeneities and truncated forms of the protein can be removed. For crystallization, a high concentration of protein is required. Vivaspin sample concentrators are disposable ultrafiltration devices designed for fast, nondenaturing concentration of biological samples. Vivaspin can also be used for desalting/buffer exchange. This study describes a two-step purification of (histidine)6-tagged MtDXR (MtDXR-[His]6 , Mr 42 000), using affinity chromatography and gel filtration. Vivaspin was used to concentrate the sample in between the two purification steps as well as for buffer exchange prior to crystallization.

to prevent protein precipitation. In the second step, Superdex™ 200 pg, prepacked in HiLoad™ 16/60 column, was used for gel filtration. The pooled fractions from gel filtration were buffer exchanged and concentrated using Vivaspin 20, MWCO 10 000 and further concentration was performed using Vivaspin 500, MWCO 10 000 intended for smaller volumes. Protein concentration was determined by measuring UV absorbance at 280 nm. The fractions were analyzed by SDS-PAGE under reducing conditions using ExcelGel™ SDS Gradient 8–18. The gel was stained with Deep Purple™ Total Protein Stain and scanned using Ettan™ DIGE Imager. Crystallization was performed using the concentrated protein by the hanging-drop vapor-diffusion technique.

Results Purification and crystallization of MtDXR-(His)6 In the first purification step, clarified lysate of E.coli expressing the recombinant protein was purified with HisTrap HP 1 ml (Fig 2). To remove unwanted E. coli proteins, a linear imidazole gradient was used for elution. Six milliliter of eluant was concentrated 2.5-fold with Vivaspin 6, MWCO 10 000 and buffer exchange was performed using Disposable PD-10 Desalting Column. Column: Sample: Binding buffer:

HisTrap HP 1 ml 16 ml of MtDXR-(His)6 (Mr 42 000) in clarified E. coli lysate 50 mM sodium phosphate, 300 mM NaCl, 10% glycerol, 10 mM imidazole, pH 8.0 50 mM sodium phosphate, 300 mM NaCl, 10% glycerol, 250 mM imidazole, pH 8.0 1 ml/min ÄKTAexplorer 100

Elution buffer:

1

Affinity chromatography HisTrap HP 1 ml

2

Concentration Vivaspin 6, MWCO 10 000

3

Buffer exchange PD-10 Desalting Columns

4

Gel filtration HiLoad 16/60 Superdex 200 pg

5

Concentration and buffer exchange Vivaspin 20 and 500, MWCO 10 000

6

Crystallization

Flow rate: System:

A280 mAU 3000 2500 2000 1500 1000 500 0

Fig 1. Workflow for the purification and crystallization of MtDXR-(His)6.

0

5

10

15

20

25

30

35

ml

Fig 2. Affinity purification of MtDXR-(His)6.

Materials and methods Figure 1 describes the workflow for the purification and crystallization of MtDXR-(His)6. All chromatography purification was performed using ÄKTAexplorer™ 100. In the first step, HisTrap™ HP 1 ml column, prepacked with Ni Sepharose™ High Performance for immobilized metal chelate chromatography, was used for affinity purification. After the affinity step, Vivaspin 6, MWCO 10 000 was used to decrease sample volume and Disposable PD-10 Desalting Column was used for buffer exchange

12

In the second purification step, concentrated eluted pool from HisTrap HP 1 ml containing MtDXR-(His)6 was loaded on HiLoad 16/60 Superdex 200 pg (Fig 3). The glycerol concentration in the pooled fractions from gel filtration decreased from 10% to 2.5% using Vivaspin 20, MWCO 10 000. Furthermore, the protein was concentrated 20-fold to 3.3 mg/ml using the same concentrator and Vivaspin 500, MWCO 10 000.


Column: Sample: Flow rate: Buffer:

HiLoad 16/60 Superdex 200 pg Concentrated eluted pool from HisTrap HP 1 ml (see Fig 2) 1 ml/min 20 mM Tris-HCl, 150 mM NaCl, 10% glycerol, pH 7.5

Innovations Forum

Protein sample preparation

SDS-PAGE analysis of the purification and concentration steps revealed high protein purity before crystallization (Fig 4). Figure 5 shows crystals obtained by the hanging-drop vapor-diffusion technique.

UV 280 nm mAu

100 80 60 40 20 0 0

20

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80

100

120

140 min

Fig 3. Gel filtration of MtDXR-(His)6. Sample from HisTrap HP 1 ml was concentrated 2.5-fold using Vivaspin 6, 10 000 MWCO, and then buffer was exchanged using Disposable PD-10 Desalting Column before loading onto the HiLoad 16/60 Superdex column.

Fig 5. Crystals of MtDXR-(His)6. were obtained using the hanging-drop vapor-diffusion technique.

Conclusions For X-ray crystallography, high protein concentration is required. Vivaspin sample concentrators provided rapid sample concentration and buffer exchange. The protein crystals obtained were suitable for 3-D structure determination.

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References

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1. Henriksson, L. M. et al. Structures of Mycobacterium tuberculosis 1-deoxy-d-xylulose-5-phosphate reductoisomerase provide new insights into catalysis. J. Biol. Chem. 282, 19905-19916 (2007).

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The protein used in this study, MtDXR-(His)6, was generously provided by L. Henriksson, Dept. of Cell and Molecular Biology, Uppsala University Biomedical Center, Uppsala, Sweden.

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Acknowlegements

Mr 97 000 66 000 45 000 30 000 20 100 14 400

Ordering information Product

28-9322-96

Vivaspin 20, MWCO 10 000*

28-9323-60

Vivaspin 500, MWCO 10 000*

28-9322-25

HisTrap HP, 5 Ă— 1 ml

17-5247-01

HiLoad 16/60 Superdex 200 pg column

17-1069-01

Disposable PD-10 Desalting Columns, 30 columns

17-0851-01

ExcelGel SDS Gradient 8–18, 6 gels

80-1255-53

Deep Purple Total Protein Stain, 5 ml (makes 1 l) 1

2

3

4

5

6

7

Fig 4. SDS-PAGE analysis of MtDXR-(His)6 after purification and concentration. Electrophoresis was performed under reducing conditions using ExcelGel SDS Gradient 8-18. The gel was stained with Deep Purple Total Protein Stain and analyzed using Ettan DIGE Imager.

Code number

Vivaspin 6, MWCO 10 000*

RPN6305

* For all molecular weight cutoffs and the different formats available, see data file 28-9356-53.

For more information about Vivaspin sample concentrators, visit www.gelifesciences.com/sampleprep

13


Label-free interaction analysis

Innovations Forum

Reliable detection and characterization of anti-drug antibodies using Biacore T100 Immunogenicity Package K. Tjärnlund GE Healthcare, Biacore, Uppsala, Sweden

Label-free, real-time protein interaction analysis is an invaluable tool for the detection and characterization of unwanted immune responses. Screening for anti-drug antibodies with Biacore™ instruments has the advantage of detecting early immune responses characterized by low affinity antibodies that interact with the drug with rapid kinetics. These immune responses can be clinically significant, but are easily missed by alternative endpoint assay-based techniques due to losses during washing procedures etc. The new Biacore T100 Immunogenicity Package from GE Healthcare offers dedicated support for immunogenicity testing by providing tools for screening, confirmation, and characterization of immune responses. It also addresses drug interference by enabling the measurement of anti-drug antibodies in the presence of excess amounts of drug.

higher sensitivity, the importance of detecting both early and late immune responses resulted in Boehringer Ingelheim implementing Biacore as their choice of immunogenicity screening platform.

Detection of anti-drug antibodies in the presence of drug Drug present in serum samples can bind to ADAs and interfere with the detection step, thereby generating false negatives. This is a general problem for immunogenicity assays, and in Biacore assays, drug interference would prevent ADAs from binding to the surface of the sensor chip (Fig 2). Increasing concentration of drug

Introduction Most biopharmaceuticals elicit some level of antibody (Ab) response against the drug, which in some cases can lead to serious adverse effects and/or loss of efficacy. Immunogenicity is therefore an important factor that must be considered in the development of new biotherapeutics, particularly due to extensive regulatory demands. The market for biotherapeutics is a rapidly growing area, and consequently, there is an increasing need to study immune responses in preclinical and clinical development.

Reliable detection of anti-drug antibodies Detection and characterization of unwanted immune responses is an important aspect of drug safety studies. Biacore T100 Immunogenicity Package can detect these immune responses that may be missed by alternative endpoint assay-based techniques, due to losses during washing procedures etc. Screening for anti-drug antibodies (ADAs) with Biacore instruments has the advantage of detecting early immune responses characterized by low affinity antibodies that interact with the drug with rapid kinetics.

Fig 2. Excess amount of drug in samples may inhibit ADAs from binding to the sensor chip surface.

Biacore T100 Immunogenicity Package addresses the issue of drug interference by enabling measurement of ADAs in the presence of excess amounts of drug. Samples are acidified to dissociate drugantibody complexes, and then neutralized just before measurement to prevent complexes from reforming (Fig 3). This enables a sensitivity of < 0.5 μg/ml Ab in the presence of 100-fold molar excess of drug. Acidification

Neutralization

Type II immune response “persistent”

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Fig 3. Acidification and neutralization allows the Biacore assay to be performed accurately in the presence of excess amount of drug. Samples are acidified to dissociate drug-antibody complexes, and then neutralized just before measurement to prevent complexes from reforming.

Biacore (ng equiv./ml)

In Figure 1, Boehringer Ingelheim compared a Biacore assay with a bridging ELISA in a phase I, multidose clinical study for a therapeutic humanized Ab. Figure 1A shows that Biacore T100 was able to detect ADAs much earlier than the ELISA in subjects displaying a type II or “persistent” immune response. In subjects displaying a type I or “transient” immune response, the Biacore assay detected several positives while the ELISA did not (Fig 1B). Although the ELISA assay formally had a

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Fig 1. A phase I, multidose clinical study for a therapeutic humanized Ab. Biacore assay (blue bars) and bridging ELISA (red bars) were used to detect Ab responses in predose serum samples from subjects receiving 12 weekly infusions of 100 mg therapeutic Ab. Detection of ADAs in subjects displaying an (A) type II “persistent” immune response and (B) type I “transient” immune response. The Biacore assay detected ADAs in early treatment samples, several weeks before ELISA detected positives. For subjects displaying a transient immune response, the ELISA did not detect a response.

14


Response - nonsp ecific background (RU)

Rat serum

Response

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Innovations Forum

80

RU 1200

Label-free interaction analysis

In a study using Rituximab, different concentrations of anti-Rituximab Abs were mixed with increasing amounts of drug (Fig 4). The detection of ADAs was inhibited as the drug concentration increased. Using the acidification and neutralization steps, the ADA response could be measured even in the presence of an excess amount of drug.

Anti-Rituximab, 0.5 µg/ml in serum

Anti-Rituximab, 0.5 µg/ml in serum, acidified and neutralized

0

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Fig 5. Rat serum bound to the sensor surface was challenged sequentially with isotype-specific reagents. The binding responses indicate the presence and relative proportions of different isotypes in the sample Ab population, and in this case, the isotype was mainly IgG2a.

50

Rituximab (µg/ml)

Fig 4. Anti-Rituximab Abs (0.5 μg/ml) were mixed with increasing amounts of Rituximab in negative human serum. Due to drug interference, the measured ADA response decreased as the Rituximab concentration increased (curve A). When anti-Rituximab Abs at 0.5 μg/ml were mixed with drug and the samples acidified and neutralized prior to measurement, the ADA response was completely recovered (curve B).

Comprehensive characterization of ADAs Biacore T100 Immunogenicity Package offers dedicated software tools for comprehensive characterization of immune responses. To characterize Ab class and subclass using Biacore systems, the antibodies in the sample are bound to the sensor surface, challenged with a series of isotype-specific reagents (typically antibodies), and the binding responses indicate the presence and relative proportions of the different Ab isotypes present. In Figure 5, rat serum was bound to the sensor surface and sequential injection of different anti-isotype reagents showed that the sample Ab population consisted mainly of IgG2a. With Biacore systems this type of assay is simplified and data is generated in real-time, whereas ELISA usually requires one microplate for each Ab isotype tested, making it more cumbersome. In addition, Biacore systems enable monitoring of Ab maturation via assessment of binding stability. The Ab population in positive samples can be characterized in terms of the binding stability to the drug on the sensor surface. ADAs usually represent a heterogeneous population with a spectrum of different properties. Usually, using a simple exponential decay to study dissociation does not properly fit the data, in part because of the heterogeneous Ab population, but also because Abs are bi- or multivalent, and avidity influences binding stability. Software tools in the Biacore T100 Immunogenicity Package can be used to characterize the immune response in terms of Ab fractions or populations with rapid and slow dissociation rates.

Conclusions Label-free, real-time Biacore assays give vital information that cannot be provided by alternative technologies, by enabling detection and characterization of low affinity Abs with rapid off-rates. Biacore T100 Immunogenicity Package provides dedicated tools for reliable detection and characterization of ADAs during preclinical and clinical development. In addition to detecting low affinity Abs often associated with an early immune response, the package addresses drug interference by enabling measurement of ADAs in the presence of excess amounts of drug. Software tools are also provided for comprehensive characterization of ADAs. Biacore T100 Immunogenicity Package, available as an optional module for Biacore T100, offers one platform that can be used throughout the immunogenicity workflow from screening to characterization of immune responses.

Acknowledgements We would like to thank Dr. U. Kunz (Boehringer Ingelheim Pharma GmbH & Co. KG, Germany) for kindly contributing data, and Dr. D. Mytych (Amgen, Inc., USA) whose group we collaborated with during the project.

Ordering information Product Biacore T100 Immunogenicity Package Includes: Biacore T100 Immunogenicity Software, License Agreement with product key, Biacore T100 Immunogenicity Handbook – including assay guidelines and recommendations

Code number 28-9423-71

For more information about the Biacore T100 Immunogenicity Package, visit www.gelifesciences.com/biacore

15


Nucleic acid purification

Innovations Forum

Comparative analysis of illustra triplePrep Kit for the isolation of genomic DNA, total RNA, and total denatured protein from a single undivided sample R. Dhulipala1; R. Bruno1; C. Cai1; K. Kapolka1; S. Lindqvist2, and M. Jiang1 1 2

GE Healthcare, Piscataway, USA GE Healthcare, Uppsala, Sweden

illustra™ triplePrep Kit enables the isolation of high quality genomic DNA, total RNA, and total denatured protein from a single undivided sample. Comparative analysis revealed higher yields of equal or higher quality than those obtained from the same input sample amounts using DNeasy™ and RNeasy™ kits (Qiagen™) and 2-D fluorescence difference gel electrophoresis (2-D DIGE). Until recently, researchers in fields such as functional genomics, molecular genetics, and biomarker studies used three separate kits to isolate DNA, RNA, and proteins for use as probes and targets in downstream applications. However, the use of divided samples could potentially skew results due to heterogeneity between different cell and tissue samples. The demand for good correlation between transcript (gene) expression, protein expression, copy number variation, and SNP detection has resulted in the development of illustra triplePrep Kit. This new kit enables the isolation of high quality genomic DNA (gDNA), RNA, and proteins from a single undivided source of tissue or cells in less than one hour. We tested the performance of illustra triplePrep Kit by using animal tissues from different organs (e.g., liver, kidney, spleen, brain, lung, and intestine) or cultured mammalian cells (e.g., HeLa, NIH-3T3, CHO-K1, and HEK-293). Analysis of data related to yields and purity showed superior performance of illustra triplePrep Kit compared to the DNeasy Blood and Tissue Kit and RNeasy Mini Kit from Qiagen and 2-D fluorescence difference gel electrophoresis (2D-DIGE), the reference method for protein isolation (1).

The procedure used for purification of DNA, RNA, and protein is described in Figure 1. Tissue or cells were lyzed in lysis buffer, loaded onto a DNA mini column, and gDNA eluted with elution buffer. Acetone was added to flowthrough containing RNA and protein and loaded onto an RNA mini column. Following RNA binding to the second column, DNase was used to remove any remaining gDNA contamination. The flowthrough from the RNA column containing only proteins was isolated by precipitation. Purity of nucleic acids was measured by NanoVue™ spectrophotometer and quality was measured using Agilent™ Bioanalyzer. Total protein yield was compared with 2-D DIGE using 2-D Quant Kit and the quality of total proteins isolated was tested by Western blotting and LC-MS.

Results Genomic DNA gDNA was isolated from 10 mg of rat liver tissue using the illustra triplePrep Kit or the DNeasy Kit. Yield, purity, and quality were compared for both samples (Fig 2). gDNA was used to amplify a 1.5 kb fragment by PCR. The resulting fragment was then sequenced to compare performance in downstream applications. Results showed higher than average yields with illustra triplePrep Kit (15.4 µg ± 0.60) compared to DNeasy Blood and Tissue Kit (9.8 µg, ± 2.10) using the same amount of input sample. gDNA isolated from both kits produced similar purities as determined by A260/A280 optical density ratios (1.90 ± 0.02). The sequenced PCR fragment generated from illustra triplePrep Kit showed higher Phred illustra triplePrep Kit

DNeasy Kit

Materials and methods DNA, total RNA, and total protein were extracted from 10 mg of rat liver tissue or 1 × 106 HeLa cells using the illustra triplePrep Kit. The performance of this kit was compared against kits used to isolate gDNA (DNeasy Blood and Tissue Kit, Qiagen), RNA (RNeasy Mini Kit, Qiagen), and protein (2-D DIGE) individually. Comparative analysis was based on sample input amount, yield, purity, speed, and ease-of-use following the manufacturers’ recommended protocols. DNA purification

RNA purification

Protein isolation

1. Sample homogenization and lysis

2. DNA binding

20 scores (744 bp) compared to DNeasy Blood and Tissue Kit (723 bp) indicating the suitability of gDNA in downstream applications such as PCR and sequencing.

Flowthrough

3. DNA wash 1

6. RNA binding

4. DNA wash 2

7. DNase (optional)

10. Protein precipitation

5. DNA elution

8. RNA wash

11. Protein wash

9. RNA elution

12. Protein resuspension

Flowthrough

Lysis 5–30 min DNA purification < 10 min

RNA purification < 10 or 20 min

Fig 1. illustra triplePrep Kit workflow.

16

Fig 2. 2% of isolated gDNA from 10 mg of rat liver analyzed on an 0.8% agarose gel shows higher yield and better quality with illustra triplePrep Kit compared with DNeasy Blood and Tissue Kit. M = molecular weight ladder.

Protein isolation < 30 min

Total RNA Total RNA was isolated from 10 mg of rat liver tissue using illustra triplePrep Kit or RNeasy Mini Kit. Yield, purity, and quality were compared for both samples (Fig 3). Both kits yielded high quality total RNA (Fig 3A). The total RNA samples were used to amplify high-, medium-, and lowexpressed 18S ribosomal RNA, cytochrome P450, and c-fos gene, respectively by real-time quantitative PCR (RT-qPCR). Similar slopes and CT values were obtained in all three cases, indicating similar performance of the kits in downstream applications (Fig 3B). Total RNA isolated from both kits produced similar quantities and purities as determined by A260/A280 ratios (data not shown).


A)

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Fig 3. Total RNA isolation from 10 mg of rat liver tissue using the illustra triplePrep Kit and RNeasy Mini Kits. (A) Analysis using Agilent Bioanalyzer revealed intact 18S and 28S ribosomal RNA bands indicating high quality RNA. (B) RT-qPCR results for amplified high-, medium-, and low-expressed 18S ribosomal RNA, cytochrome P450, and c-fos gene.

[nt]

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Innovations Forum

Nucleic acid purification

A)

2

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Total protein

Conclusions illustra triplePrep Kit provides rapid isolation of gDNA, RNA, and protein from single undivided samples. This kit enables researchers to correlate DNA, RNA, and protein data from the same sample and generate data related to gene expression, protein expression, and genomic analysis from precious limited samples such as biopsies, archived tissues, and tumors. Comparative analysis with DNeasy Blood and Tissue Kit, RNeasy Mini Kit, and the 2-D DIGE reference method showed higher yields of DNA, RNA, and total protein, respectively with equal or better quality enabling successful applications in PCR, RT-qPCR, sequencing, Western blotting, and LC-MS.

References 1. Henkel, C. et al. Changes of the hepatic proteome in murine models for toxically induced fibrogenesis and sclerosing. Proteomics 6, 6538–6548 (2006). 2. Aljanabi, S.M. and Martinez, I. Universal and rapid salt-extraction of high quality genomic DNA for PCR- based techniques Nucl. Acids Res. 25, 4692–4693 (1997). 3. Vogelstein, B. and Gillespie, D. Preparative and analytical purification of DNA from agarose. Proc. Natl. Acad. Sci. USA 76, 615 (1979).

HeLa cells

C)

Rat liver

100 90 80

Relative abundance

Proteins were isolated from the second flowthrough after isolating gDNA and total RNA following the illustra triplePrep protocol. Yield was similar for the both illustra triplePrep Kit and 2D-DIGE reference method (Fig 4A). Western blotting using appropriate antibodies was used to detect β-actin or GAPDH proteins (Fig 4B). The peptides were separated by nanoRPC on Ettan™ MDLC coupled to a Finnigan LTQ™ Linear Ion Trap mass spectrometer (Thermo Fisher Scientific Inc.). The LC-MS data was evaluated using DeCyder™ MS Differential Analysis Software. Protein samples from the illustra triplePrep Kit extraction were also analyzed by LC-MS for peptide profiling and showed average representation of 3115 ± 176 peptides (Fig 4C).

70 60

39.04 30.96

20.78

40.48

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44.47

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55.06 59.7561.03 64.18

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10.6515.0616.24

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54.14

48.06

22.11 26.61

10

73.00

10 15 20 25 30 35 40 45 50 55 60 65 70 75

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Fig 4. (A) Protein isolated from the illustra triplePrep Kit and 2-D DIGE reference method produced similar yields. (B) Western blotting analysis of proteins shows the presence of β-actin and GAPDH. (C) The base peak ion chromatogram of LC-MS shows a wide peptide distribution and signal intensity from protein isolated by illustra triplePrep Kit.

Ordering information Product

Code number

illustra triplePrep Kit, 50 preps

28-9425-44

2-D Quant Kit

80-6483-56

For more information on our range of protein and nucleic acid sample preparation products, visit www.gelifesciences.com/illustra

4. Sambrook, J. and Russell, D. W., Molecular Cloning: A Laboratory Manual, chapter 6, Cold Spring Harbor Laboratory Press, New York (2001).

17


GE & Science Prize 2008

Understanding a minimal DNA-segregating machine Reconstitution of a plasmid spindle shows how three components together accomplish the task of DNA segregation E. C. Garner Systems Biology, Harvard Medical School, Boston, MA, USA A current challenge in biology is to bridge the gap between the parts and the whole, to reconcile the biochemical properties of individual proteins with the emergent behaviors of multipart molecular machines. In theory, if every kinetic rate constant for every interaction were measured, we could gain a complete mechanistic understanding of a biological process. Because the number of required measurements scales with the number of components, most complex biological systems present difficult challenges for this approach. However, when quantitative insight is matched with the proper system, the goal of elucidating the biochemical basis of emergent behaviors can be realized. For example, to gain a molecular-level understanding of DNA segregation, a fundamental, mesoscale process, I turned to a system that has been forced to be minimal and self-contained during its evolution: low-copy bacterial plasmids. To maintain their inheritance against the fitness costs imposed by their extra metabolic burden, these exogenous elements have evolved minimal, self-contained, DNA segregation machines. At a minimum, a DNA-segregating system needs to accomplish three tasks. First, it must count the copies of DNA to know when to initiate segregation. Second, it must exert directional force to propel these DNA copies away from each other. Third, this system must be spatially aware of the cellular geometry so that the DNA is propelled toward each eventual daughter cell. For the Escherichia coli R1 drug resistance plasmid, all of these tasks are conferred by the par operon, which constructs a mitotic spindle out of only three components (1). The centromeric sequence parC contains 10 repeats that are bound by the adapter protein ParR. This ParR/parC complex interacts with ParM, a distant actin homolog that polymerizes into helical filaments (2). In my thesis work, I determined how these three components interact to accomplish the systems-level task of DNA segregation. To elucidate this mechanism of plasmid segregation, I needed to understand the nature of ParM polymerization, how the ParR/parC complex affected ParM filaments, and finally, how these three components interact to push the plasmids to the poles of the cell. First, I conducted a complete characterization of ParM assembly dynamics, measuring all available rate constants for this polymer (3). Although ParM is a structural homolog of eukaryotic actin, I found three distinct differences between these polymers. First, ParM nucleates filaments at a rate 300 times as fast as actin. Second, unlike any other previously observed polymer, ParM shows no polarity as it grows at equal rates at each end of the filament. The most striking difference between ParM and actin is that ParM exhibits dynamic instability, the stochastic switching between states of growth and rapid disassembly (4). Previously, this behavior had only been observed for eukaryotic microtubules. ParMâ&#x20AC;&#x2122;s dynamic instability is driven by ATP hydrolysis, as ADP-bound ParM filaments display a much higher dissociation rate than the ATP-bound filaments. The combination of dynamic instability and rapid nucleation causes solutions of ParM to consist of a population of short, dynamic filaments that nucleate and turn over throughout the volume (Fig 1). This unstable,

18

transient nature appears at odds with the formation of a rigid forcegenerating mitotic spindle, suggesting that the ParR/parC complex must alter ParM filament dynamics. I tested this hypothesis by reconstituting the par system from purified components (5), combining parC-conjugated beads with ParR and fluorescently labeled ParM. Isolated parC beads displayed short, dynamic asters of ParM emanating from their surface, as if they were searching the surrounding volume. When two parC beads came into close contact, stable bundles of filaments formed between the beads. These filaments then elongated at a constant rate, pushing the beads in opposite directions.

Fig 1. Dynamic instability and bipolar stabilization drive plasmid segregation. The ParR/parC complex can capture cytoplasmic ParM filaments. Filament ends bound by ParR/parC are stabilized (blue) while unbound filaments (red) and destined to undergo catastrophe. A productive spindle is formed when both ends are bound by ParR/parC. Turnover of the unattached, background filaments provides the energy differential (arrows) to power spindle elongation.

Creating fiducial marks on these filaments demonstrated that they elongate at equal rates at each bead surface, indicating that new monomers are added into the spindle through a process of insertional polymerization at the ParR/parC complex. This was the first in vitro reconstitution of a DNA-segregating system from purified components. The fact that we observed long, stable ParM filaments only between pairs of beads indicated that the filaments are stabilized against catastrophic disassembly when bound at both ends by ParR/parC. When these spindles were severed with laser irradiation, these filaments would rapidly depolymerize. This bipolar stabilization provides an intrinsic counting mechanism, as productive and sustained filament elongation only occurs between parC pairs. I then tested whether these spindles could locate the ends of a volume by confining them in microfabricated channels of various shapes. These spindles aligned with and elongated along the long axis of these spaces, indicating that this simple system is sufficient to find the long axis of a cell.


GE & Science Prize 2008

In addition to demonstrating that these three components are necessary and sufficient to generate the emergent behaviors required for DNA segregation—counting, force generation, and spatial awareness—this work also elucidated the biophysical relationship between dynamic instability and force generation. By conducting spindle assembly assays at varying concentrations of ParM, I found that filaments bound at each end by ParR/parC behave as if the entire filament is composed of the higher-affinity, ATP-bound polymer. In essence, this stabilizes the bound filaments to a lower energetic level than the unbound filaments that turn over in solution (see Fig 1). This indicates that the dynamic instability of the unattached filaments provides the monomer excess that powers the elongation of the stabilized ParR/parC attached filaments. These studies provide a framework for understanding the essential principles of DNA segregation. Furthermore, they demonstrate that biology can solve complex tasks with a surprisingly small number of components. This minimal solution to DNA segregation is not an isolated case; many low-copy plasmids have independently converged on three-component systems by co-opting a variety of different polymers from their hosts (6–9). Kinetic dissection of a range of these self-contained mitotic machines may uncover the differing solutions that biology has evolved to ensure genetic inheritance and thus broaden our understanding of this fundamental biological process.

References 1. K. Gerdes, S. Molin, J. Mol. Biol. 190, 269 (1986). 2. J. Møller-Jensen, R. B. Jensen, J. Löwe, K. Gerdes, EMBO J. 21, 3119 (2002). 3. E. C. Garner, C. S. Campbell, R. D. Mullins, Science 306, 1021 (2004). 4. T. Mitchison, M. Kirschner, Nature 312, 237 (1984). 5. E. C. Garner, C. S. Campbell, D. B. Weibel, R. D. Mullins, Science 315, 1270 (2007). 6. S. Austin, A. Abeles, J. Mol. Biol. 169, 353 (1983). 7. T. Ogura, S. Hiraga, Cell 32, 351 (1983). 8. E. Becker et al., EMBO J. 25, 5919 (2006). 9. R. A. Larsen et al., Genes Dev. 21, 1340 (2007). Readers may view, browse, and/or download this material for temporary copying purposes only, provided these uses are for noncommercial personal purposes. Except as provided by law, this material may not be further reproduced, distributed, transmitted, modified, adapted, performed, displayed, published, or sold in whole or in part, without prior written permission from the publisher.

First published in Science 322, 1486–11487 (2008). Reprinted with permission from AAAS. To view the winning essays and to apply for the 2009 awards, visit www.sciencemag.org/feature/data/prizes/ge

19


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Discovery Matters 9-2009  

Discovery Matters Magazine

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