Austria VWR International GmbH Graumanngasse 7 1150 Wien Tel.: 01 97 002 0 Fax: 01 97 002 600 E-mail: [email protected]

Belgium VWR International bvba Researchpark Haasrode 2020 Geldenaaksebaan 464 3001 Leuven Tel.: 016 385 011 Fax: 016 385 385 E-mail: [email protected]

Denmark VWR - Bie & Berntsen Transformervej 8 2730 Herlev Tel.: 43 86 87 88 Fax: 43 86 87 90 E-mail: [email protected]

Finland VWR International Oy Valimotie 9 00380 Helsinki Tel.: 09 80 45 51 Fax: 09 80 45 52 00 E-mail: [email protected]

France VWR International S.A.S. Le Périgares – Bâtiment B 201, rue Carnot 94126 Fontenay-sous-Bois cedex Tel.: 0 825 02 30 30 (0,15 EUR TTC/min) Fax: 0 825 02 30 35 (0,15 EUR TTC/min) E-mail: [email protected]

Germany VWR International GmbH Hilpertstrasse 20a D - 64295 Darmstadt Tel.: 0180 570 20 00* Fax: 0180 570 22 22* E-mail: [email protected] *0,14 €/Min. aus d. dt. Festnetz, Mobilfunk max. 0,42 €/Min.

Hungary VWR International Kft. Simon László u. 4. 4034 Debrecen Tel.: (52) 521-130 Fax: (52) 470-069 E-mail: [email protected]

Ireland / Northern Ireland

Spain

VWR International Ltd / VWR International (Northern Ireland) Ltd Orion Business Campus Northwest Business Park Ballycoolin Dublin 15 Tel.: 01 88 22 222 Fax: 01 88 22 333 E-mail: [email protected]

Sweden

Italy VWR International s.r.l. Via Stephenson 94 20157 Milano (MI) Tel.: 02 332 03 11 Fax: 800 152 999 E-mail: [email protected]

The Netherlands VWR International B.V. Postbus 8198 1005 AD Amsterdam Tel.: 020 4808 400 Fax: 020 4808 480 E-mail: [email protected]

Norway VWR International AS Haavard Martinsens vei 30 0978 Oslo Tel.: 02290 Fax: 815 00 940 E-mail: [email protected]

Poland

VWR International Eurolab S.L. C/ Tecnología 5-17 A-7 Llinars Park 08450 - Llinars del Vallès Barcelona Tel.: 902 222 897 Fax: 902 430 657 E-mail: [email protected]

VWR International AB Fagerstagatan 18a 163 94 Stockholm Tel.: 08 621 34 00 Fax: 08 621 34 66 E-mail: [email protected]

Switzerland VWR International AG Lerzenstrasse 16/18 8953 Dietikon Tel.: 044 745 13 13 Fax: 044 745 13 10 E-mail: [email protected]

UK VWR International Ltd Customer Service Centre Hunter Boulevard Magna Park Lutterworth Leicestershire LE17 4XN Tel.: 0800 22 33 44 Fax: 01455 55 85 86 E-mail: [email protected]

Labart Sp. z o.o. A VWR International Company Limbowa 5 80-175 Gdansk Tel.: 058 32 38 210 Fax. 058 32 38 205 E-mail: [email protected]

Portugal VWR International - Material de Laboratório, Lda Edifício Neopark Av. Tomás Ribeiro, 43- 3 D 2790-221 Carnaxide Tel.: 21 3600 770 Fax: 21 3600 798/9 E-mail: [email protected]

Go to vwr.com for the latest news, special offers and details of your local VWR distributor. EN-072011-VTDSVZ

Issue 27 Autumn 2011

the market source for life science

I Page 10 Real-time PCR analysis of surfactin gene expression I Page 25 Quantitative Western blotting utilising new horizontal gel system I Page 30 Batch and fed batch cultivation of different mammalian cell lines

the market source for life science

editorial Hope you had a great summer and enjoy being back at work with this autumn edition of the BioMarke Magazine. New layout, new partner and lots of applications to help you in your life science work. Joining us: Polyplus Transfection, a biotechnology company that develops innovative solutions for in vitro and in vivo delivery of nucleic acids in reasearch, bioproduction and therapeutics. Our long standing members have plenty to contribute too with innovations in some interesting areas such as stem cell therapy with integrated systems for the expansion of Mesenchymal

stem cells from BD or transfection techniques with jetPRIME™, a powerful and versatile transfection reagent developed by Polyplus to deliver DNA and siRNA into adherent cells ... Also included with this issue is the VWRbioMarke Shop, a tabloid filled with special offers on key products – often linked to the magazine articles. This year everyone is feeling the pinch of rising prices and spending restrictions so make sure that you have a look through this flyer to help you get the most of your budget! Very best regards The VWRBioMarke team

CONTENTS genomics NEW BTX Hybrimune System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 The qScript™ microRNA Quantification System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 - 6 Techne® thermal cycler performance testing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 White PCR disposables for quantitative real-time PCR from BRAND . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 - 9 Real-time PCR analysis of surfactin gene expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 - 11 DNA/RNA extraction from formalin fixed, paraffin embedded tissues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 - 13 EvaGreen® dye - the next generation of DNA binding dye . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 - 15 New AgilePulse in vivo system DNA vaccine development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16 Versatile thermal cycler product range . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 DNA and siRNA transfection with jetPRIME™ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18 - 19 Editor

PROTEOMICS Determining the most effective Dialysis MWCO for protein purification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 - 21 Pall Life Sciences - centrifugal devices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22 - 23 OligoClear™ - removing oligonucleotide contaminations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24 Quantitative Western blotting with Amersham™ ECL™ Gel system & Amersham™ ECL™ Prime . . . . . . . 25 - 27 Clean, simple and rapid purification of antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28 - 29

CELL biology Batch and fed batch cultivation in the BIOSTAT® Aplus bioreactors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Drug screening using Corning® Osteo Assay Surface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Thermo Scientific Nunclon™ Vita™ surface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ChillProtec®: New protective medium for cold storage of cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Building a reliable foundation for stem cell research . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Petakas´ internal environment reflects the O2 concentration of tissues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

30 - 32 33 - 35 36 - 37 38 - 40 41 - 43 44 - 47

VWR International Europe bvba Researchpark Haasrode 2020 Geldenaaksebaan 464 3001 Leuven Belgium

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Layout and typesetting Marketing Services VWR

Printing

Stork, Bruchsal, Germany No part of this publication may be reproduced or copied without prior permission by writing of VWR International Europe.

Run

80 120 copies Publication date: September 2011 Due to the high sales volume of promoted articles some items may be temporarily out of stock - VWR Terms and Conditions of Sale apply.

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I VWR International I VWRbioMarke Issue 27 I September 2011

genomics

For more information on these products contact your local VWR sales office, send an e-mail to [email protected] or visit our website www.vwr.com

NEW BTX Hybrimune System Large volume hybridoma production

Human antibodies for Iimunotherapy development generated via a human B cell hybridoma technology.

Human specific monoclonal antibodies, mAbs, have great potential for safe and targeted therapy for many diseases. Ideally, therapeutic Abs are produced in human B cells for optimal human effector functions and with limited immunogenicity. This can be done with hybridomas, generated by fusing antigenspecific B cells with an immortal B cell line (myeloma). This work describes hybridoma production for the generation of fully human therapeutic mAbs, using electrofusion methods to align and electroporate the cells with high fusion efficiency and high cell viability.

Conclusions Electrofusion proved to be an efficient method used to generate hybridoma lines secreting mAbs with high binding specificity and biological activity. One mAb with strong neutralising activity against human granulocyte–macrophage colony stimulating factor was identified.

Results 1. Hybridomas were produced by electrofusion of ex vivo immunised primary human B cells with K6H6 B5 cells at a 1:1 lymphocyte:K6H6 B5 ratio. On average 60% of seeded wells contained viable hybridoma cells, and 1200 clones were screened after each immunisation. 2. ELISA assay demonstrated specificity of human GM-CSF mAbs generated from the hybridomas (Figure 1). 3. A cell-based assay demonstrated the biological activity of human mAbs against GM-CSF. G9 and G10 mAb inhibit growth of human GM-CSF-dependent TF1 cells (Figure 2). 4. Hybridomas were stable, showing homogeneous retention of Ig production after 60 generations.

Figure 1. Antigen panel demonstrates antigen-specific human mAbs from hybridomas. Three GM-CSF-specific human mAbs, E5, G7, and E10, reacted with human GM-CSF and none of the other antigens in the panel. The controls reacted as expected: Anti-TT mAb reacted only to TT, 215, a murine mAb specific to human GM-CSF, reacted to both hGM-CSFand mGM-CSF, and anti-IL-3 reacted only to IL-3.

Figure 2. Biological activity of human mAbs against GM-CSF. A cell-based assay was carried out to determine the level of GM-CSF neutralisation (% of inhibition) mediated by G9 and E10 mAbs. Human GM-CSF-dependent TF1 cells were incubated in the presence of 0,1 ng ml GM-CSF. No inhibition of growth was observed when normal isotype control IgG was included in the reaction, regardless o the concentration used. Both E10 and G9 mAbs neutralised GM-CSF activity.

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the market source for life science

qScript™ microRNA Quantification System: Superior validation of microRNA profiling data Ralph Hector, Sistemic UK and David Cheo, Quanta Biosciences

Data obtained from high throughput gene expression profiling platforms is often validated using real-time RT-qPCR. Recently the application of these technologies has been expanded to include microRNAs. We have compared the performance of the qScript™ microRNA Quantification System from Quanta Biosciences to another commercially available RT-qPCR system. The qScript™ microRNA Quantification System was found to be more sensitive than the competitor’s system providing a higher degree of confidence in the accurate detection and quantification of microRNAs.

Introduction MicroRNAs (miRNAs) are a unique class of short (~22 nucleotide) non coding RNA molecules that are conserved in nature and function primarily to silence gene expression (1). Targets for miRNAs consist of specific complementary sequences within the 3’-untranslated regions of messenger RNA (mRNA) transcripts. When miRNAs hybridise to their targets they downregulate gene expression by enhancing mRNA degradation or inhibition of protein synthesis. Since each of the more than 1200 human miRNAs can potentially regulate multiple mRNAs, as much as 60% of the human transcriptome may be regulated by miRNAs as predicted by recent bioinformatic analyses (2). Acting in concert with complex regulatory

networks, miRNAs participate in the control of basic cellular processes such as growth, differentiation and development. Mutation and dysregulation of miRNAs have been implicated in the pathogenesis of many human diseases making miRNA important targets for disease diagnostics and therapeutics. High throughput expression profiling of miRNAs is commonly performed using microarray and next generation sequencing platforms. Typically, profiling data is subsequently validated using real-time RTqPCR. There are many commercially available kits designed to quantify the expression level of miRNAs. Results from different miRNA quantification systems can vary greatly depending on multiple factors including the

Figure 1. Comparison of the qScript™ microRNA Quantification System from Quanta Biosciences (blue lines) to a competitors system (red lines). A: Amplification data from hsa-miR-125b representing a log-fold dilution series of cDNA. The qScript™ microRNA Quantification System detected hsa-miR-125b with more than ten fold higher sensitivity than the competitors system at each input amount of cDNA. B: Amplification data from hsa-miR-1247 representing qPCR amplification of plus-PAP (solid lines) and no-PAP (broken lines) cDNA synthesis reactions. The qScript™ microRNA Quantification System detected hsa-miR-1247 with 64 fold (5 Cts) higher sensitivity and more than 128 fold (7 Cts) difference from the no-PAP control compared to a 2 Ct difference in the competitors system. Insets: Dissociation (melt curve) analysis of SYBR Green RT-qPCR products shows specific amplification of a specific product from the qScript™ microRNA Quantification System as noted by the predominance of a single peak at the expected Tm. The melt curve from Lower cDNA yield from the competitor’s system indicated lower yield and the presence of non specific amplification product.

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I VWR International I VWRbioMarke Issue 27 I September 2011

genomics

For more information on these products contact your local VWR sales office, send an e-mail to [email protected] or visit our website www.vwr.com

amount of RNA available and the abundance of miRNAs in each sample. When RNA samples are limiting or miRNAs are present in low abundance, accurate detection and measurement of miRNA expression levels can be challenging. We have compared the performance of the qScript™ microRNA Quantification System from Quanta Biosciences to a competitor’s miRNA assay system. The qScript™ microRNA Quantification System was determined to be more sensitive in the detection and quantification of miRNAs and provided a superior validation of miRNA expression profiling data.

Materials and methods Total RNA isolation and QC Cells were cultured in 6 well plates in supplemented medium until 80% confluent. Total RNA was isolated from each sample using the miRCURY Cell & Plant RNA isolation kit (Exiqon, Vedbaek, Denmark) following the manufacturer’s instructions. Total RNA was quantified using an ND-1000 (NanoDrop, Wilmington, USA) and RNA integrity was checked using an Agilent 2100 Bioanalyzer

(Agilent, Santa Clara, USA), following the manufacturer’s instructions. All total RNA samples had an RNA integrity number of ≥8,9.

Microarray Total RNA from each sample (100 ng) was labelled using the miRNA Complete Labeling and Hyb Kit (Agilent), following the manufacturer’s instructions, and hybridised to Human miRNA Microarrays V3 (Agilent). Arrays were washed and scanned using the Agilent Microarray Scanner, following the manufacturer’s instructions. Feature Extraction (Agilent) was used to QC the arrays and retrieve miRNA probe fluorescence data from the scanned image. Feature Extraction confirmed that each microarray was of high QC standards.

Real-time RT-qPCR Specific miRNAs were analysed using two miRNA assay systems and the results of relative quantification on the MX3005P™ Real-Time PCR System (Stratagene, Santa Clara, USA) were compared. For the qScript™ microRNA Quantification System (Quanta Biosciences, Gaithersburg, USA), total RNA from each

Figure 2. Two independent sample sets were used to compare RT-qPCR data to microarray data. In the first set (left panel) hsa-miR-15a was measured in RNA samples from three liver-derived cell lines (A, B and C). In the second set (right panel) hsa-let-7a was measured in RNA samples from three adipose-derived stem cells (D, E and F). qPCR data (green and red bars) was plotted against microarray data (black bars) for each specific. For hsa-miR-15, samples B and C were expressed at a significantly higher level (p20 kD; 100 kD being the optimal MWCO for IgG purification. Refering to the MWCO retention profile curves, the 50 kD and 100 kD MWCO membranes effectively eliminate Vitamin B12 (the 1/100X low MW contaminant) in 24 hours; while the 20 KD MWCO would require approximately 36 hours. Only the 100 kD MWCO effectively eliminates Cytochrome C (the 1/10X MW contaminant), however it requires 2 - 3 days for completion. The 20 and 50 kD MWCO’s are simply ineffective in eliminating Cytochrome C.

General Rules for Protein Purification by Dialysis: 1. Dialysis requires that the target protein or macromolecule is at least 10X larger, ideally 100X larger, than the contaminants to be removed. 2. The most effective MWCO for protein purification is the highest MWCO just below the size of the protein to be retained. 3. To achieve purification in 1 – 2 days, the membrane MWCO should be at least 100X larger than the contaminant MW. 4. To achieve, purification in 2 - 3, the membrane MWCO should be at least 10X larger than the contaminant MW.

Retention in membrane MWCO’s 20 kD 50 kD 100 kD 99.0 % 99.0 % 99.0 % 99.0 % 98.8 % 98.8 % 99.0 % 98.2 % 97.5 % 96.0 % 93.5 % 90.0 % 92.0 % 85.0 % 72.5 % 88.0 % 76.0 % 28.0 % 76.1 % 62.4 % 42.7 % 52.5 % 36.5 % 18.8 % 12.4 % 2.5 % 0.6 %

Table 1

VWR International I VWRbioMarke Issue 27 I September 2011 I

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the market source for life science

Pall Life Sciences - centrifugal devices Exclusive to VWR

There are 3 generic applications for Ultra-Filtration: 1. Concentration.

Pall Life Sciences centrifugal devices simplify many common nucleic acid and protein sample preparation procedures. These devices provide efficient concentration and salt removal of samples from 50 μl to 60 ml in just minutes.

Ultra-Filtration (UF) is a membrane separation technique based on selection by molecular size, although other factors, such as molecule shape and charge, can also play a role. Molecules larger than the membrane pores in the UF membrane will be retained at the surface of the membrane while solvent and smaller solute molecules will freely pass. This molecular exclusion at the UF membrane surface leads to concentration of the protein solute in the retained fraction (termed the retentate) and can be recovered from above the membrane.

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Ultra-Filtration is a very convenient method for the concentration of dilute protein or DNA/RNA samples. It is gentle (does not shear DNA as large as 100 Kb or cause loss of enzymatic activity in proteins) and very efficient (typically >90% recovery).

2. Desalting and buffer exchange (diafiltration).

Ultra-Filtration provides a convenient and efficient way to remove or exchange salts, remove detergents, separate free from bound molecules, remove low molecular weight components, or rapidly change the ionic or pH environment.

3. Fractionation.

Ultra-Filtration will not accomplish a sharp separation of two molecules with similar molecular weights. The molecules to be separated should differ by at least one order of magnitude (10X) in size for effective separation. Fractionation is effective in applications, such as the preparation of protein-free filtrates, the separation of unbound or unincorporated label from DNA and protein samples, and the purification of PCR products from synthesis reactions.

Nanosep® and Nanosep® MF centrifugal devices Simple, reliable concentrating and desalting of 50 to 500 μl samples • Ensures rapid processing of samples. • Typical recoveries are >90% - available with low protein binding • Omega™, Bio-Inert®, and GHP membranes. • A wide range of MWCOs, colour coded for easy identification • Constructed of low binding polypropylene. • Ultrasonically welded seals prevent bypass or seal failure • Fits standard centrifuge rotors that accept 1.5 ml tubes

Centrifugal devices can replace traditional separation techniques, such as column chromatography, preparative electrophoresis, alcohol or salt precipitation, dialysis, and gradient centrifugation, when performing the following: • Protein or nucleic acid concentration • Desalting • Buffer exchange • Deproteination of biological samples • Fractionation of protein mixtures • Separation of primers from PCR products • Separation of labelled nucleic acids or proteins from unincorporated nucleotides • Virus concentration or removal • Clarification of cell lysates and tissue homogenates

I VWR International I VWRbioMarke Issue 27 I September 2011

Pall Life Sciences offer a complete range of Ultra-Filtration devices suitable for various sample volumes. These centrifugal devices are specifically engineered to provide faster flow rates and easier handling. Each device is available with either Omega™ Ultra-Filtraion membrane or 0,2 and 0,45 m Supor ® membrane for microfiltration applications. Devices have a colour coded label to provide quick, easy identification of molecular weight cut-off and pore size.

Proteomics

For more information on these products contact your local VWR sales office, send an e-mail to [email protected] or visit our website www.vwr.com

Microsep™ Advance centrifugal devices

Macrosep™ Advance centrifugal devices

Jumbosep™ centrifugal devices

Precise, quick recovery of microlitre volumes of concentrate from starting volumes up to 5.0 ml • High recovery - achieves 50X concentration and >90% recovery in just minutes • Features deadstop to prevent samples from spinning to dryness • Versatile Omega™ membrane is available in a variety of MWCOs • Colour coded and laser etched for easy identification.

Quickly concentrates up to 20 ml of biological sample without valuable sample loss • Rapidly concentrates 20 ml sample volumes to 0.5 ml • Provides high recoveries, typically >90%. • Low protein-binding Omega™ membrane and polypropylene housing minimises loss due to non specific binding • Versatile Omega™ membrane is available in a variety of MWCOs • Built-in deadstop prevents spinning to dryness • Colour coded for easy identification

Convenient and reliable concentration, purification, and diafiltration of 15 to 60 ml biological samples • Concentrates 60 ml sample volumes to 5 ml in 30 minutes • Provides high recoveries, typically >90 %. • Low protein-binding Omega™ membrane and polysulphone housing minimise losses due to non specific binding • Versatile Omega™ membrane is available in a variety of MWCOs • Colour coded for easy identification • Built-in deadstop prevents spinning to dryness • Unique sealing mechanism prevents retentate leakage and filtrate contamination • Economical - sample reservoir and filtrate

See BioMarkeShop for special offer! Nanosep® centrifugal devices with Omega™ membrane Description Pk Cat. No. 24 516-8490 10K, blue 100 516-8491 24 516-8501 30K, red 100 516-8502 24 516-8519 100K, clear 100 516-8520

Macrosep™ Advance centrifugal devices with Omega™ membrane Description Pk Cat. No. 10K, blue 6 516-0357 24 516-0358 30K, red 6 516-0360 24 516-0361 100K, clear 6 516-0363 24 516-0364

Microsep™ Advance centrifugal devices with Omega™ membrane Description Pk Cat. No. 24 516-0372 10K, blue 100 516-0373 24 516-0374 30K, red 100 516-0375 24 516-0376 100K, clear 100 516-0377

Jumbosep™ centrifugal device starter kits Description Pk 10K starter kit, blue 4 30K starter kit, red 4 100K starter kit, clear 4

Cat. No. 516-8159 516-8160 516-8161

For more information about Pall’s complete range of Ultra-filtration devices, including complete product portfolio and application notes, please visit www.pall.com/lab or speak to your local VWR sales office.

VWR International I VWRbioMarke Issue 27 I September 2011 I

23

the market source for life science

OligoClear™ enables you to remove oligonucleotide contaminations from your molecular diagnostic application Nowadays sensitivity of DNA amplification technology is so high that the slightest contamination with oligonucleotide sequences can disrupt your production work flow. This leads to costly incoming goods inspections, a need for increased stock and possible difficulties during scale-up at a later stage of the process. While GMP oligonucleotides are manufactured to be safe in human application they are not necessarily designed to avoid the slightest cross contamination that may interfere with PCR amplification and the cost and delivery time could be hardly bigger. Since its foundation in 1995 Thermo Fisher Scientific’s Custom Biopolymer site is well known for the synthesis of finest oligonucleotides, each one is delivered purified by true reverse phase HPLC as standard. Proactively preventing contaminations always results in a smoother and more cost effective production. Through close collaboration with our customers in the field of molecular diagnostics we developed highly customised services to meet specific requirements in terms of purity, reliability and cost.

Optional services can be added to the basic service to meet your requirements. For example, dedicated HLPC columns, pipettes or syringes for your project or for each oligo, specific decontamination procedures, sterile filtration, desalting, solubilisation, high precision concentration measurement etc…

Understanding that every customers demands are different, we are now offering an OligoClear™ “à la carte menu” of services to help you removing contamination threats from your production process. The service always includes specifically trained employees, synthesis on dedicated equipment, chemicals batch tracking, analysis by mass spectrometry and comprehensive documentation.

OligoClear™ optional services can include:

See BioMarkeShop for special offer!

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I VWR International I VWRbioMarke Issue 27 I September 2011

To discuss your needs and design your “a la carte” OligoClear™ menu, contact our experienced scientists at [email protected].

- Proprietary, reserved HPLC columns - Reserved utility objects (syringes, bulbs, spatulas) - Special decontamination procedures - Desalting by size exclusion column - Special OD measurement for increased precision - Sterile filtration of final product

Proteomics

For more information on these products contact your local VWR sales office, send an e-mail to [email protected] or visit our website www.vwr.com

Quantitative Western blotting utilising new horizontal design Amersham™ ECL™ Gel system and Amersham™ ECL™ Prime Maria Winkvist, Susanne Grimsby and Karin Söderquist GE Healthcare Bio-sciences AB, Björkgatan, 30, SE-751 84 Uppsala, Sweden.

GE Healthcare pioneered the ECL™ approach to Western blotting under the Amersham™ brand. Traditional Western blotting with chemiluminescence is a very well established technique used to study proteins from a wide variety of sources.

See BioMarkeShop for special offer!

The technique is used throughout life sciences from basic research to medical diagnostic applications. For some Western blotting applications, a “yes or no” answer is enough, but the addition of quantitative data will for many applications be beneficial. To obtain good quantitative Western blotting data there is a demand on broad linear dynamic range, high signal-to-noise ratio, stability of signals and normalisation of the total protein amount that was loaded on the respective gel lane. Moreover, high sensitivity will have an impact on the linear dynamic range as well as the possibility to detect low abundant proteins, which is critical for many Western blotting applications. Here we demonstrate the use of a new ECL™ reagent, Amersham™ ECL™ Prime and new Amersham™ ECL™ precast gels, in a number of typical Western blotting applications. The results demonstrate that Amersham™ ECL™ Prime reaches limits of detection enabling analysis of low abundant proteins, that signals are very stable over time and cover a broad dynamic range. These features make Amersham™ ECL™ Prime highly suitable for accurate quantitative Western blotting. New Amersham™ ECL™ precast gels ensure reliable and reproducible results. The novel horizontal design makes gel handling and sample loading very easy, which enables new users to successfully perform high quality protein electrophoresis and Western blotting.

Methods Co-IP Western blotting

TAK1 was immunoprecipitated in untreated and treated PC3U cell lysates using anti-TAK1 mouse IgG1 monoclonal antibodies and Protein G Mag Sepharose™. Unbound proteins were washed away and TAK1 with its interacting proteins were eluted from the beads. The eluate was applied to Western blotting for detection of the interacting protein TAB 1.

P38 phosphorylation assay

HEK 293T cells were exposed to TGF-ß. SDS-PAGE of cell lysates on Amersham™ ECL™ Gel was performed according to standard procedure and was used to evaluate levels of p38 and phosphorylated p38 (pp38) by Western blotting. Relative quantitation was performed after normalisation by comparison to levels of the housekeeping protein GAPDH.

Amersham™ ECL™ Prime Western blotting

Western blotting was performed according to the standard procedure and protocol supplied with the reagent using Hybond™ P membrane (PVDF), optimised blocking solution and specific antibodies at optimised concentrations.

Detection and imaging

Amersham™ ECL™ Prime detection reagent was added to the membrane (Amersham™ Hybond™ P (PVDF) and the chemiluminescent signal was captured using ImageQuant™ LAS 4000 mini Biomolecular Imager.

Analysis

Images were analysed using ImageQuant™ TL 7.0 software.

Signal stability

The signal duration was followed up to three hours after addition of Amersham™ ECL™ Prime. Signals were captured every 10 minutes using the same exposure time at every time point (3 minutes). The signal intensity for the band corresponding to 0,31 ng protein (transferrin) was analysed and the remaining signal intensity was compared to the initial signal intensity.

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the market source for life science

Results High sensitivity of ECL™ Prime enables detection of low abundant proteins With a limit of detection down to low picogram levels, Amersham™ ECL™ Prime demonstrates its usefulness for

applications ranging from confirmatory protein detection with demand on high sensitivity as well as detection of low

abundant proteins and post-translational modifications.

B

A

Figure 1. Monitoring of a plasma purification process with a total protein stain Deep Purple™ and confirmation of IgG content with Amersham™ ECL™ Prime Western blotting (A). Western blotting detection of transferrin in a twofold dilution series using Amersham™ ECL™ Prime (B).

Broad linear dynamic range is needed for reliable quantitation A Western blotting system has a linear dynamic range when the detected signals are directly related to the amount of protein on the blot. A broad linear dynamic range allows the comparison of weak and strong bands on the same blot and requires that (1) high end signals are not saturated and that (2) A

the lower limit of detection is as low as possible. A lack of linearity, caused by saturation of strong signals, will lead to underestimation of protein abundances at the high end of the scale (Figure 2). By using Amersham™ ECL™ Prime and ImageQuant™ LAS 4000 mini system proteins can be detected

with a linear dynamic range of three orders of magnitude starting on low picogram levels as demonstrated here using transferrin as an example. This data demonstrates the usefulness of the system when quantitation across a wide level of protein concentrations is required.

B

Figure 2. Western blotting detection of transferrin in a twofold dilution series using Amersham™ ECL™ Prime, image obtained with ImageQuant™ LAS 4000 mini system (A). An illustration of broad linear dynamic range and the consequence of a saturated signal (B).

In-lane normalisation is a prerequisite for accurate quantitation Normalisation with a housekeeping protein can correct for uneven sample loading, uneven transfer to membrane, etc. This makes it a necessary prerequisite

A

for accurate quantitation. The best way to do this is to relate the protein of interest to an unregulated housekeeping protein like actin (Figure 3).

C

Figure 3. A schematic illustration of how to relate your protein of interest to a housekeeping protein (A). A Coimmunoprecipitation of endogenous TAB1 with TAK1 in cell lysates from untreated and TGF-ß treated PC3U cells. The Coimmunoprecipitation was performed with Protein G Mag Sepharose and detection with Amersham™ ECL™ Prime and ImageQuant LAS 4000 mini (B). Detection of different levels of phosphorylated STAT3 (pSTAT3) in 5 different HeLa cell lysates. Relative quantitation of pSTAT3 levels after normalisation to actin for correction of uneven sample amount (C).

B

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In this way, it is possible to compare levels of specific protein between lanes even when the total protein loads are not identical.

I VWR International I VWRbioMarke Issue 27 I September 2011

Proteomics

For more information on these products contact your local VWR sales office, send an e-mail to [email protected] or visit our website www.vwr.com

Stability of signal and efficient use of antibodies is convenient and cost saving The stability of signal over time and the ability to dilute the antibodies allows flexibility, convenience and efficient use of precious antibodies. (Figure 4)

Figure 4. Amersham™ ECL™ Prime signal duration was followed 3 hours after the addition of reagent by capturing images every 30 minutes with the same exposure time (3 minutes). The signal intensity for the band corresponding to 0.31 ng protein (transferrin) is analysed and the remaining signal intensity was compared to the initial signal intensity (A). Comparison of Western blotting detection performance using highly diluted antibodies. ß-catenin was detected in twofold dilution series of NIH 3T3 whole cell lysate using primary antibody dilutions ranging from 1:3000 to 1:10 000 and secondary antibody at 1:30 000 or 1:50 000 (B).

Quantitating post-translational modifications p38 is a mitogen-activated protein kinase involved in cell differentiation and apoptosis and is regulated by phosphorylation. Here, HEK 293T cells

were exposed to TGF-ß. After SDSPAGE of cell lysates on Amersham™ ECL™ Gel, Western blotting was performed to evaluate levels of p38 and phosphorylated p38 (pp38) over time. Relative quantitation was performed after normalisation by comparison to levels of the housekeeping protein GAPDH. (Figure 5) Figure 5. Quantitative Western blotting analysis of p38 and pp38 following stimulation of HEK 293T cells with TGF-ß. p38 responded to stimulation with TGF-ß by phosphorylation after 30 minutes. Note that pp38 signals in isolation would indicate a peak in phosphorylation levels after 120 minutes. Normalisation by comparison with GAPDH signals shows that this is not the case and that phosphorylation of p38 is maximal after 30 minutes and then remains phosphorylated in the presence of TGF-ß.

Figure 6. The new Amersham™ ECL™ Gel system, consisting of the Gel Box electrophoresis unit and a selection of ready-to-use pre-cast gels (10%, 12%, 4 - 12%, 8 - 16%, and 4 - 20% acrylamide, 10, 15 or 2 sample wells) has a novel horizontal design. This simplifies handling, especially the sample loading, reduces buffer consumption (190 ml per run) and significantly reduces the risk of leakage. It utilises Tris/Glycine buffers. The gels can be run as denaturing gels using the standard Laemmli system or as native gels.

Conclusions The high sensitivity of Amersham™ ECL™ Prime combined with the broad linear dynamic range of CCD-based imagers, such as ImageQuant™ LAS 4000 series, enables relative quantitation of target proteins with confidence. This is demonstrated with several typical Western blotting applications, with a limit of detection on the low picogram level and a broad linear dynamic range. Moreover, stability of the signal over time and possibilities to dilute antibodies more allows convenient handling, enables repeated exposures and efficient use of precious antibodies. Together with the new Amersham™ ECL™ precast gels consistent separation with high resolution was observed. The new horizontal design enables easy operation and sample loading.

Acknowledgements Professor Marene Landström Umeå University, Ludwig Institute for Cancer Research Uppsala is acknowledged for kindly providing cell lysates for p38 and Co-IP experiments and valuable discussions.

Description Amersham™ ECL™ Gel 10%, 10 wells Amersham™ ECL™ Gel 10%, 15 wells Amersham™ ECL™ Gel 12%, 10 wells Amersham™ ECL™ Gel 12%, 15 wells Amersham™ ECL™ Gel 4 - 12%, 10 wells Amersham™ ECL™ Gel 4 - 12%, 15 wells Amersham™ ECL™ Gel 8 - 16%, 10 wells Amersham™ ECL™ Gel 8 - 16%, 15 wells Amersham™ ECL™ Gel 4 - 20%, 10 wells Amersham™ ECL™ Gel 4 - 20%, 15 wells Amersham ECL Gel Box

Pk 10 gels 10 gels 10 gels 10 gels 10 gels 10 gels 10 gels 10 gels 10 gels 10 gels 1

Cat. No. GEHE28-9898-04 GEHE28-9901-55 GEHE28-9898-05 GEHE28-9901-56 GEHE28-9898-06 GEHE28-9901-57 GEHE28-9898-07 GEHE28-9901-58 GEHE28-9901-54 GEHE28-9901-59 GEHE28-9906-08

VWR International I VWRbioMarke Issue 27 I September 2011 I

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the market source for life science

Clean, simple and rapid purification of antibodies Purify IgG from serum in under 15 minutes with Pearl™ antibody purification resin

G-Biosciences’ new Pearl™ IgG purification resin functional groups bind to the majority of proteins present in serum, ascites and tissue culture supernatants, allowing the IgG to rapidly pass through and be collected in the flowthrough fraction. The main advantage of Pearl™ IgG purification resin is that IgG antibodies are not purified by a bind and release mechanism, which is rapid and extremely gently on the IgG molecules. This results in highly pure and active IgG antibody molecules.

Another advantage is that it is compatible with a wider variety of different species and subclasses of antibodies, compared to the expensive, bind and release, Protein A and Protein G resins (Table 1). Pearl™ IgG purification resin enables antibodies purified from the resin are ready for use in downstream applications, including immunoassays or subsequent conjugation/ labelling reactions. The mild elution conditions mean that further purification is not required to neutralise or desalt the sample, a common requirement with bind and release resins, such as Protein A or Protein G.

Products In addition to Pearl™ IgG purification resin, G-Biosciences offers four complete kits that utilise the Pearl™ IgG purification resin.

Pearl™ IgG purification (spin format): For the purification of up to 25 mg antibody in