Thermo Scientific Open Biosystems Custom Antibody and Proteomics Services

Transcription

1 Thermo Scientific Open Biosystems Custom Antibody and Proteomics Services Available from the Thermo Scientific Pierce Protein Research Products Portfolio

2 Antibody Services Production Plan Summary Antibodies can be generated in a variety of different species using more than one dozen specialized protocols. We can produce full-length recombinant proteins, protein fragments or peptides to be used as antigens or you can send antigens to us for immunization. Recombinant Protein Antibodies Look to us for high-quality customized services for your protein characterization and detection needs. Services include custom peptide synthesis, custom protein expression and custom polyclonal and monoclonal antibody development. Whether you need high-quality peptides for enzymatic research or robust antibodies for quantitative protein measurement, discover what a completely integrated proteomics service provider can do for you. Our suite of world-class custom services includes: Custom polyclonal antibodies Custom hybridoma (monoclonal) development Custom peptide synthesis Custom protein expression Custom monoclonal production Purified recombinant proteins can be excellent immunogens for antibody production. Send us 1.5 mg of your protein, and we will immunize into a selected protocol to generate antibodies. Any protein weighing more than 9 kda is sufficient to produce an immune response without conjugation. Haptens weighing less than 9 kda will need to be conjugated to a carrier protein before immunization. Rabbit polyclonal packages start as low as US$499. If you do not have the purified protein available, we can subclone from a cdna and express it for you (see the Protein Expression section on page 9 for more information). We have the world s largest collection of cdnas to choose from, saving you even more time and money. A minimum of 500 µg of protein and the antibody is supplied for projects for which we express the protein. Packages include: Immunization protocol Pre-bleeds and production bleeds Test-aliquots for convenience ELISA titration data on all bleeds Online project tracking OLAW- and USDA-approved animal protocols Peptide Antibodies Synthetic peptide antibodies can be designed to specifically perform in your intended assay application, maximizing specificity and cross-reactivity with orthologous sequences. Advanced antigen design and targeted antigen display provide an unmatched benefit in the development of highutility antibodies that work on both the peptide and on the endogenous protein target. Peptide antibodies include: Synthetic peptide up to 20 residues Protein analysis and antigenic peptide design with Thermo Scientific Antigen Profiler Software Peptide synthesis and purification HPLC and mass spectrometry on peptides Targeted antigen display conjugation of peptide to KLH (BSA, OVA, THY and RSA are also available) Immunization protocols in rabbits, chickens, guinea pigs or goats ELISA titration of all bleeds Optional peptide affinity purification 1 For more information, or to download product instructions, visit

3 Antibody Services Protein Modification Antibodies Selective populations of polyclonal antibodies can be isolated from larger total clonal populations to study modified forms of proteins. This includes modifications such as protein cleavage, mutations, polymorphisms and phosphorylation. A series of specialized protocols are available to generate reagents to meet these needs. Each protocol includes a three-step affinity purification in which antibodies to the modified target are enriched and cross-reactive antibodies to the unmodified target are removed through the use of affinity depletion columns. The final purified product is delivered along with quantitative ELISA results that measure the preference of the antibodies to the modified versus unmodified form. Some of the more common modifications we generate antibodies against are listed in the adjacent table. Conjugation and Antigen Presentation Thermo Scientific Targeted Antigen Display Technology is a key feature of our antibody development strategy. Up to five different chemistries are available to pinpoint specific linkage sites on the peptide of interest Specific side groups are targeted based on their benefit in crosslinking to mimic assay conditions Epitope exposure is maximized within structural motifs and orientation to the termini Peptides and proteins are confined structurally through the site-specific introduction of chemical linkers and helper peptides Protein modifications. phosphorylation sumoylation myristolation acetylation polymorphisms drug binding ubiquitination glycosylation isoforms splice variants ligand binding prenylation protein cleavage (neo-epitopes) mutations species cross-reactivity Novel methodologies are used for presenting antigens in conformationally relevant contexts that mimic the presentation of the native protein in the assay of interest. This technology combined with the Antigen Profiler Software enables generation of superior assay-specific antibodies. Available carrier proteins. KLH: BSA: OVA: THY: MAP: keyhole lympet hemocyanin bovine serum albumin ovalbumin thyroglobulin multiple antigen peptide (no conjugation necessary) 2 Polyclonal Antibody Hosts and Immunization Protocols Rabbits are the preferred host species used to generate polyclonal antibodies, but alternate species are offered. Only SPF-certified New Zealand white rabbits are used for rabbit antibody generation. All animals are contained in barrier-enclosed, climate-controlled facilities that have been approved by the NIH (OLAW) and the USDA, and the facilities have AAALAC accreditation. Alternate species protocols are available upon request. If you re not sure which species is most appropriate for your applications, our scientists can help you make this decision. Alternate species choices include: Chicken Mice/rats Sheep/goats Guinea pigs You can also submit your own protocols for antibody production, which we will submit to the IACUC panel for approval. At right: Additional options are available to meet your needs, including affinity purification, antibody labeling, additional animals and customized immunization protocols. We also offer protocols for antibodies raised in goat, chicken, mouse, rat and guinea pig. Polyclonal antibody packages. Selected rabbit polyclonal packages 2 Rabbit 90-day protocol 2 specific pathogen free (SPF) rabbits 4 immunizations/animal 1 pre-immune and 4 production bleeds/animal ELISA titers on all bleeds Deliverables: 5-10 mg peptide (if applicable) ELISA data Pre-immune control aliquots ml serum 2 Rabbit 70-day protocol 2 SPF rabbits 4 immunizations/animal 1 pre-immune and 3 production bleeds/animal ELISA titers on all bleeds Deliverables: 5-10 mg peptide (if peptide ordered) ELISA data Pre-immune control aliquots ml serum For more information, or to download product instructions, visit Options Peptide design synthesis and conjugation Customer-supplied antigen Customer-supplied conjugated antigen Design, synthesis, conjugation and co-injection of two peptides Protein modification specific (phosphorylation, acetylation, splice variants, etc.) with affinity-purified, negative absorbed antibody Peptide design synthesis and conjugation Customer-supplied antigen Customer-supplied conjugated antigen Design, synthesis, conjugation and co-injection of two peptides

4 Antibody Services, continued Assay Formatting By accounting for the conformational changes that occur in proteins under different assay conditions, antibody binding can be optimized for specific applications of interest. This is a crucial step to a project s success. Types of assay-specific antigen design protocols. Immunohistochemistry (IHC) Immunofluorescence (IF) Western Blotting Immunoprecipitation (IP) ELISA Immunocytochemistry (ICC) Fluorescence activated cell sorting (FACS) Chromatin IP (ChIP) Competitive inhibition (Antagonists) Continuation/Extension Protocols After the completion of the initial immunization protocol, animals can be either terminally bled, terminated with no further action or placed on one of a number of extension protocols. Extension protocols have been specifically designed for increasing immune responses to stubborn antigens or for maintaining high titers for long-term antibody collection. Polyclonal Antibody Purification A variety of different purification strategies are offered, depending on the needs for the antibody; however, peptide affinity chromatography is the most popular choice. Affinity purification selects for only the immunogen-specific antibodies removing unwanted pre-immune and cross-reactive immunoglobulins. Protein A, Protein G and ammonium sulfate precipitation are also available to isolate total antibody fractions. All affinity purifications performed involve at least two different purification steps. For more complicated purifications that involve the negative adsorption of antibodies against unmodified forms of peptides or negative control cell lysates (e.g., phospho-antibodies), up to four different purification steps may be involved. Continuation protocols. Description Standard monthly extension High-dosage extension Aggressive extension Holding animals Terminal bleeds Antibody purification options. Description Service One boost, one bleed and maintenance of animal for one month One boost with higher dosage antigen, bleed and maintenance for one month Two boosts seven days apart at higher dosages followed by bleeding and maintenance One-month maintenance options with no boosting or bleeding Exsanguination on request Service Total IgG purification Ammonium sulfate precipitation Protein A/G purification Peptide affinity purification Affinity depletion Highly specific protocol to isolate all IgG from serum or supernatant Immobilized peptide on affinity resin following by ph elution Used to enrich for antibodies to specific modifications (e.g., phos) A number of activated resins are used for immobilizing molecules, depending on the conjugation method used and project strategy. Purification Quality Assurance The final step of the purification is to evaluate and qualityassure the process by indirect ELISA against the peptide or protein bound to the solid-phase and by measuring antibody concentration by the Thermo Scientific Pierce BCA Protein Assay. The protein concentration reading measures the total amount of protein and, therefore, the total amount of antiimmunogen antibody in the eluent. In the ELISA, two pieces of data are examined the eluent and the serum flow-through. Concentration and yield data are supplied with the final delivery of the purified antibodies, while the quantitative ELISA data is sent approximately one week after shipment. Affinity Purified ELISA Titers Assay Description The following are your affinity purified ELISA results. This is the final step of the purification process, and it is used to evaluate and quality-assure the purification protocol. Purified antibody is titered by indirect ELISA against the peptide or protein bound to the solid-phase to measure the reactivity of the antibodies after elution (the eluent) and the amount of antibody remaining in serum (the flow-through). The eluent titer indicates the minimum concentration at which your antibody can effectively detect your antigen. The flow-through titer represents the reactivity of the antibodies remaining in the serum after it has been passed through the column. Any antibody that does not stick to the column should be found in this fraction. Volume Conc. Total Project # Customer (ml) (mg/ml) (mg) Titer Flow-through ng A John Doe AF301, AF302 A John Doe ng AF303, AF304 A John Doe ng AF305, AF306 The titer results are obtained by first calculating the concentration of antibody eluent through absorbance (OD280/OD205). The next step is to titrate the antibody 1: 1 across the plate based upon the concentration starting at an arbitrary 10 µg/ml. The concentration at which the antibody signal drops below the detection threshold is where the eluent titer is set and reported. The lower the titer is the more sensitive antibodies. The flow-through titer is determined by taking the serum that passed through the column and performing 1: 1 serial dilutions with the neat serum across the plate. This value can be directly compared with the crude titers by multiplying it times two. The affinity purified antibody titers are reported differently than crude serum titers, because the exact amount of specific anti-peptide or anti-protein antibody in the solution is known. The eluent titer is reported as a concentration, which enables the reporting of much more quantitative values versus the qualitative dilution value with crude serum. If your antibodies have been negatively adsorbed against any related protein or peptide, such as the non-phosphorylated analog peptide, these results will be reported as well showing the preference for the modified versus unmodified forms. P: (888) F: (256) support@openbiosystems.com

5 Design and Produce Better Antibodies The Thermo Scientific Open Biosystems Custom Antibody and Biomarker Development Service leverages our experience in making more than 18,500 antibodies to peptides and recombinant proteins, our proprietary antigen design tools, and targeted antigen display to produce more robust antibodies that perform better in your targeted assays. Custom Antibody Options Recombinant Protein Polyclonal Anti-Peptide Polyclonal Monospecific Polyclonal Phospho-Specific Polyclonal Anti-Peptide Monoclonal Recombinant Protein Monoclonal Advanced Antigen Design with Antigen Profiler Software Peptide Synthesis NA NA Depleting Peptide or Control Peptide NA NA Optional NA Protein Expression Optional NA NA NA NA Animal Immunization Protocol ELISA Titration Affinity Purification and Depletion Optional Optional NA NA Fusions and Cloning NA NA NA NA Monoclonal Production and Purification NA NA NA NA Optional Optional Complete Online Project Tracking 3

6 Custom Monoclonal Antibodies Hybridoma Development Protocol Details Monoclonal antibodies can offer significant advantages for specificity and long-term antibody reproducibility but, as with polyclonals, the final application(s) must be heavily considered. Custom Monoclonal Antibodies Novel immunization strategies, antigen design and screening technologies are essential for the successful generation of monoclonal hybridoma cells lines. Our immunization protocols take advantage of using multiple strains of mice along with altered immunization routes, combinations of ligand carriers and dosing strategies to produce as diverse of a collective monoclonal antibody population as possible. This polyclonal utility is essential because the diversity of clones for immortalization is a major limiting factor in traditional monoclonal generation. Antigens must be carefully designed, especially when dealing with peptides, to generate a robust reagent that is able to recognize conformational changes of the native protein in assay conditions. Hybridoma development advantages: Vary dosage, carriers and routes Immunize multiple stains of mice Clone up to 10 positive hybrids Assay-specific hybrid selection (capture, IP, etc.) Antigen-specific ELISAs 5-Mouse Hybridoma Protocol Recommended for highly purified recombinant proteins for which antibodies will be used primarily in Western blotting or immunohistochemistry (IHC). Details: 1. Immunization of five Swiss Webster (SW) mice over 11-week protocol 2. Four boosts, three test-bleeds, ELISA titration 3. Fusion of best two mice by ELISA and/or customer evaluation of crude antiserum 4. Screening by ELISA-based assay 5. Evaluation of positive sups by customer 6. Expansion and freeze down of parental cell lines 7. Delivery of up to five best parental cell lines as frozen stocks 10-Mouse Hybridoma Protocol Recommended for highly purified recombinant proteins and peptide-conjugates for which antibodies will be used primarily in Western blotting or IHC, but are also needed for multiple applications in which epitope structure is extremely important (IP, capture, etc.). Details: 1. Immunization of five SW and five Balb/c (10 mice total) over 11-week protocol 2. Four boosts, three test-bleeds, ELISA titration of all bleeds 3. Fusion of best three mice by ELISA and/or customer evaluation of crude antiserum 4. Screening by ELISA-based assay (negative screen included) 5. Evaluation of positive sups by customer 6. Expansion and freeze down of parental cell lines 7. Delivery of up to 10 best parental cell lines as frozen stocks 15-Mouse Hybridoma Protocol Recommended for peptide-conjugates, compounds and small antigens for which antibodies will be used primarily in Western blotting or IHC, but are also needed for multiple applications where epitope structure is extremely important (IP, capture, etc.). Details: 1. Immunization of 10 SW and five Balb/c (15 mice total) over 11-week protocol 2. Four boosts, three test-bleeds, ELISA titration of all bleeds 3. Fusion of best five mice by ELISA and/or customer evaluation of crude antiserum 4. Screening by ELISA-based assay (negative screen included) 5. Evaluation of positive sups by customer 6. Expansion and freeze down of parental cell lines 7. Isotype confirmation of clones 8. Growth optimization and rate determination as related to antibody expression 9. Delivery of up to 10 best parental cell lines as frozen stocks Total production time is approximately four to six months. Development is broken down into three phases that defer costs at each step. For each protocol, additional subcloning and monoclonal production options are available for an additional charge. 4 4 For For more more information, or to or download to product product instructions, visit visit

7 Monoclonal antibodies. Standard Monoclonal Packages Phase 1 and 2 Phase 3 Deliverables 5 Balb/c Mouse Hybridoma Development 10 (5 BALB/c and 5 SW) Mouse Hybridoma Development 15 (5 BALB/c and 10 SW) Mouse Hybridoma Development Immunization and ELISA. Terminate or continue based on results. Select mice for fusion spleen cells with myeloma P3x63Ag8.653, evaluate hybridomas by ELISA, and select and freeze selected cell lines. Up to 2 fusions performed. Supernatants and 5 frozen cell lines shipped. Up to 3 fusions performed. Supernatants and 5 frozen cell lines shipped. Up to 5 fusions performed. Supernatants and 10 frozen cell lines shipped. Additional options including antigenic peptide synthesis, purification and conjugation, cell culture antibody production in roller bottles or T-flasks, ascites production, and antibody purification services are available. Monoclonal antibodies. Standard Monoclonal Packages Phase 1 and 2 Phase 3 Recommended Antigens 5 Balb/c Mouse Hybridoma Development Recombinant proteins or protein fragments 10 (5 BALB/c and 5 SW) Mouse Hybridoma Development 15 (5 BALB/c and 10 SW) Mouse Hybridoma Development Immunization and ELISA. Terminate or continue based on results. Select mice for fusion spleen cells with myeloma P3x63Ag8.653, evaluate hybridomas by ELISA, and select and freeze selected cell lines. Recombinant protein fragments or peptides Peptides or compounds Additional options including antigenic peptide synthesis, purification and conjugation, cell culture antibody production in roller bottles or T-flasks, ascites production, and antibody purification services are available. Hybridoma Subcloning At your request, positive parental hybridomas can be subcloned through limiting dilution cloning to obtain daughter cell lines. Subcloning is important for the long-term stability of clones and to ensure that cells are truly monoclonal and maintain clonality over their production life. We offer two options for limited dilution cloning. Up to five daughter cell lines are provided after subcloning. Daughter cell lines may all react identically. Cell viability is also confirmed through freeze-thaw. Subcloning options. Single-cycle cloning Multi-cycle complete clonality cloning Single limiting-dilution cloning to pick out consistent producers with confirmed isotype and production. Multiple rounds of limited dilution cloning until all wells are growing at the same rate, excreting antibody at the same concentration and have the same confirmed isotype. Monoclonal production: In vivo and in vitro options In vitro options include T-flask or roller-bottles Five available strains for ascites production Protein A/G or anti-immunoglobulin purification available Antibody labeling (biotin, HRP, PE, AP, FITC) Applications testing Troubleshooting (clonality, production yield, stability) To order, To order, call call Outside Outside the United the United States, States, contact contact your your local local branch branch office office or distributor. 5 5

8 Custom Peptide Services Custom Peptides Every peptide synthesis is a challenge of its own. During the last 12 years, our team has accumulated tremendous expertise through tens of thousands of successful custom peptide syntheses. By carefully listening to our worldwide customer base, we constantly adapt our product offering to meet or exceed your needs. Custom Peptide Synthesis Services We offer best-in-class custom peptide synthesis capabilities with a wide-range of labels, modifications, scales, purities and delivery time options to meet your research needs. Our never-ending commitment to quality and customer support enables you to focus on your experiments while leaving synthesis details to us. Applications: Antibody development Epitope mapping FRET Immunoassay Assay standards Ligands Enzymatic assays Agonist Co-precipitation Synthesis options We offer a large number of synthesis options. Choose from the standard scales and purities listed. Scales and purities. 5 mg 10 mg 20 mg 50 mg 100 mg > 100 mg Crude Crude Crude Crude Crude Crude >70% >70% >70% >70% >70% >70% >80% >80% >80% >80% >80% >80% >90% >90% >90% >90% >90% >90% >95% >95% >95% >95% >95% >95% >98% >98% >98% >98% >98% >98% The intended application will dictate the purity needed. For antibody development and immunoassays, we recommend a purity range of 70-90%. If you plan to use peptides for any quantitative assay, higher purities are typically required (>95%) with the optional AAA. Crude purity is suitable for any screening application such as epitope mapping. If you have a specific question about which purity and scale is appropriate for your experiment, please contact us for assistance. Fastest manufacturing time A proven record of quality Close to 100% synthesis success rates Purity you can trust A team of experts always ready to assist you quickly and efficiently An up-to-date and extensive choice of modifications Day after day this partnership will help you to achieve the best results, faster. Fastest manufacturing time In today s proteomic research, fast delivery time is a necessity. Every day our teams use a systematic approach of practical process improvement (PPI) to continuously improve our processes. In fact, we ship 75% of peptides within 10 days of order. A proven record of quality Our quality management system has been certified according to ISO9001 since For our collaborators, it is more than a certificate, it is a way of life. Every day, demanding customers from all over the world rely on our products. Close to 100% synthesis success rates Peptide synthesis results are partly dependent on the sequence, length and modification requested. The knowledge and experience of our chemists are essential to success. If it is possible, we will do it. Purity you can trust We deliver our peptides in purity grades ranging from crude to >99%. Every peptide is fully documented, including mass spectrometry and analytical HPLC certificates. We always ensure the sequence and the purity of peptides delivered. A team of experts You will always find an experienced chemist on our team ready to listen to your needs and to provide technical assistance whenever needed. Thermo Scientific HeavyPeptide Kits Basic and AQUA Grade for relative and absolute quantification of proteins We routinely synthesize peptides labelled with 13 C and 15 N isotopes. These HeavyPeptides Kits are extremely useful for quantifying your protein by mass spectroscopy. Visit for more details. 6 6 For For more more information, or to or download to product product instructions, visit visit

9 An up-to-date and extensive choice of modifications We update our portfolio continuously so we are able to provide new molecules required by scientific developments. If the modification you need is not listed, please contact us we can develop customized solutions for your specific requirements. Quality assurance. MADLI-TOF mass analysis Electrospray mass analysis (ES) Vapor-sealed bottle (nitrogen) LCMS HPLC AAA Modifications and labels available. Acetylation of N-terminus Fluorescein (FITC/5-FAM) (Ac-NH-) Acetyl-lysine Formic acid Aldehyde Glycosylation (Mannose, Galactose) 6-amino hexanoic Hydroxy proline acid (Ahx) Amidation of C-terminus (-CONH 2 ) Isotopically labeled amino acids (with 2 H, 13 C, 15 N) Amino benzoic acid Keyhole limpet hemocyanin (KLH) Beta alanine Mercaptopropionic acid Bovine serum Methoxy-coumarin-acetic acid (MCA) albumin (BSA) Biotin Methyl lysine Caproic acid Multiple antigen peptides (MAP) CBZ (Z) Myristic acid Citrulline 3-nitro tyrosine Conjugation (BSA, KLH) Norleucine (Nle) Conjugation Octanoic acid (oligonucleotides) Coumarin Palmitoic acid Custom FRET peptides Phosphorylation of Ser, Thr and Tyr Cyclization via termini Pyro glutamic acid (Pyro-E) or disulfidebridge D-amino acids Rhodamine B Dabcyl Rhodamine 110 Dabsyl Spacer Dansyl Special amino acids (D-amino acids, other amino acids) Dimethyl lysine Stearic acid Dinitrophenyl (DNP) Sulfurylation of Ser, Thr and Tyr Thermo Scientific Tetramethylrhodamine (TAMRA) DyLight Dyes Dyomics Dyes Texas Red Dye EDANS Farnesyl Other dyes or modifications on request ISO9001 quality measurement All peptides are quality monitored according to ISO9001 standards and analyzed by mass spectrometry and liquid chromatography prior to shipping to ensure target quality and purity. The most sensitive MALDI-TOF and/or electrospray (ES) are used to detect as little as 0.1 daltons size differences. Stringent elution gradient specifications and the highest quality columns are used to represent the greatest confidence in the purity determination measurement reported so you can be sure of the samples you use in assay. We offer amino acid analysis for peptides for end-users who need to determine molar concentrations for peptides in highly analytical assays. Delivery Turnaround time is key for accurately scheduling experiments. Our capabilities and throughput enable us to deliver every peptide within the shortest possible timeframe. Project managers will keep you up-to-date on all orders. For the highest priority projects, we have developed Priority Peptides, a dedicated production process. This process provides the fastest possible delivery times for peptides while maintaining our commitment to the highest quality. Priority peptide delivery time (in weeks). Scale Purity Crude >70% >80% >90% >95% >98% 2 mg mg mg mg mg mg mg mg Ordering Ordering is easy, Online quote forms or printable fax forms are available at: To order, To order, call call Outside Outside the United the United States, States, contact contact your your local local branch branch office office or distributor. 7 7

10 Thermo Scientific Custom HeavyPeptide Service Protein quantitation by mass spectrometry One of the key challenges in proteomics is the quantitation of proteins at very low concentrations in complex protein mixtures. The HeavyPeptide Solution is based on the well established isotopic dilution technique. HeavyPeptide Kits are the ultimate choice for specific, accurate and sensitive protein quantitation. As one of the leading manufacturers of mass spectrometers, many of our customers confront these challenges daily. We were also the first company to install a pilot research center dedicated to biomarker discovery (BRIMS) in Boston, MA. Amino acid Mass difference to standard AA Isotope composition Alanine/A + 4 Da U- 13 C3, 98%; 15 N, 98% Argenine/R + 10 Da U- 13 C6, 98%; 15 N4,98% Isoleucine/I + 7 Da U- 13 C6, 98%; 15 N, 98% Leucine/L + 7 Da U- 13 C6, 98%; 15 N, 98% Lysine/K + 8 Da U- 13 C6, 98%; 15 N2,98% Phenylalanine/F + 10 Da U- 13 C9, 98%; 15 N, 98% Proline/P + 6 Da U- 13 C5, 98%; 15 N, 98% Valine/V + 6 Da U- 13 C5, 98%; 15 N, 98% Other amino acids on request Carefully listening to customers led us to develop a fast, economical and easy-to-use solution: Custom HeavyPeptide Basic and Thermo Scientific AQUA Kits. For more information, please send an to abs@thermofisher.com using the code BROHP07. Modified Thermo Scientific HeavyPeptide for Quantitation of Phosphorylated Proteins Heavy peptides can be prepared with covalent modifications (e.g., phosphorylation, methylation, acetylation, etc). These are chemically identical to the naturally occurring post-translational modifications. Heavy peptides represent one of the most powerful tools for relative and absolute quantitation of post-translational modified proteins by LC-MS. Advantages: Relative or absolute quantitation Higher accuracy, sensitivity and specificity Easy-to-order custom-made heavy peptides Quantitation of low-abundance proteins Economically viable for high-throughput screening Applications: Biomarker discovery and validation Pharmacokinetics Metabolomics Clinical biochemistry (drug and metabolite monitoring) Anti-doping testing Protein expression Validation of sirna experiments Cell signalling profiling Allergen quantification Native Protein Phosphorylated Protein COOH H2N COOH H2N phos H 2 N-HL SASPSDNVYL R-COOH H 2 N-HL SASP (ps)dnvyl R-COOH A signature peptide is identified and sequenced Heavy Protein COOH H 2 N H 2 N-HL SASPSDNVYL R-COOH H 2 N-HL SASPSDNVYL* R-COOH Figure 1. Heavy peptide quantitation. 8 8 For For more more information, or to or download to product product instructions, visit visit

11 Biomarker Development and Protein Expression Services Thermo Scientific Product Name Here Custom Protein Expression We offer bacterial and mammalian protein expression services as part of our antibody development platform. Protein antigens are essential starting components for many antibody applications such as EIA and other specific quantitative protein measurement assays. Use the Thermo Scientific Open Biosystems Gene Search Tool to find your gene(s) of interest, and let us do the rest. Coding regions are subcloned into suitable expression vectors and optimized for protein expression. Protein Expression and Antibody Biomarker Development Bringing it all together a comprehensive platform for protein detection. We offer a complete platform for antibody-based biomarker development that uses cutting-edge antigen design and antibody screening tools. Take advantage of our peptide synthesis, protein expression, polyclonal and or monoclonal antibody development, and antibody production services to generate robust reagents that are designed to perform in your specific assay application. Comprehensive antigen design with Antigen Profiler Software Peptide synthesis or protein expression Targeted antigen display (conjugation and antigen manipulation) Polyclonal and/or monoclonal development Antibody production and purification Service Description Time plex subcloning Subclone gene of interest in 2 weeks plex vector with c-terminal epitope tags DNA prep Prepare plasmid DNA 1 week by maxiprep Particle Generate 10 6 lentiviral 1 week production particles Transduction Transduce CHO or HEK cells Stable cells Select for stable cell lines 2 weeks using puromycin Expression testing Overexpression testing for positive bands 1 week Protein confirmation Scale up Protein purification By Western blot with supplied antibody or with epitope tag 10-L wave bag production (~ mg protein) Chelation and FPLC 1 week 4 weeks 2 weeks To order, call To order, call or Outside the United States, contact your local branch office or distributor. 9

12 Antigen Design Software Advanced Protein Analysis Protein analysis algorithms identify active sites, signal peptides, splice variants and more than one dozen additional features, including: M odified residues: Acetylation Amidation Hydroxylation Phosphorylation Glycosylation Methylation Polymorphisms Mutations DNA binding sites Transmembrane regions Antigen Design and Presentation with Thermo Scientific Antigen Profiler Software These regions are extremely important to avoid when generating antibodies because antibodies will fail to bind if the endogenous form contains them, while the designed antigen does not. The power of bioinformatics We offer the most advanced software available to analyze proteins and design antigens for antibody generation. We have combined years of experience, literature review and the power of bioinformatics into the industry s most advanced antigen design application. The latest version of Antigen Profiler Software incorporates added features such as data mining for known and predicted protein characteristics, a sequence viewer, and automated BLAST analysis. Let us design the best antigenic peptide or protein fragment for your antibody production projects. Highlights: Examine comparative antigenicity based upon actual in vivo titer data for more than 13,500 peptide antibodies Incorporate important structural motifs that are proven sites for producing high-binding antibodies Built-in peptide tutorial for synthesis and solubility Specificity determination through curated BLAST analysis Receive a detailed report highlighting design results and protein features How does it work? Visit where you will be asked for antibody performance criteria that are used in the design process. After you submit project information into the profiler interface, our highly trained project managers will analyze your submission and then correspond directly with you to refine data based on input and requirements. An algorithm that captures antigenic determinants using mathematical relationship mapping is used. A web crawler mines data from websites, pulling defined features and known protein characteristics to build feature map and assign key details to sequence content. Output graphically displays protein(s) characteristics with selected antigens highlighted and automated interactive BLAST data. Optimal Peptide Design Algorithms Figure 1. Analysis of a complete protein highlights signal peptides, structural motifs and modified residues. Our peptide design application uses a proprietary set of algorithms to produce a variety of peptides that optimize synthesis success, solubility and antigenicity. Most importantly, each peptide is compared with our curated BLAST databases of more than two dozen species to limit cross-reactivity while improving overall antibody specificity. Designing peptides with Antigen Profiler Software puts you in control: Specify desirable cross-reactivity Target post-translational modifications Avoid epitopes unavailable due to structural constraints Eliminate resynthesis times with optimized synthesis design features Figure 2. Automated BLAST analysis compares desirable peptides across dozens of species, increasing your antibody specificity. 10 For more information, or to download product instructions, visit

13 Track and Manage Your Projects Online Online project tracking with Open Project Polyclonal antibody projects may be tracked online through a secure access point that enables you to view project schedules, provide animal instructions and gather technical data such as ELISA titers. View project and animal information Filter and sort projects and animals Download project and animal information Submit animal or purification instructions Dedicated technical and customer support We provide dedicated project managers to work with you from the beginning to the end of your project and ensure the highest level of services. Learn more about Open Biosystems Products and Services today at or contact a project manager at abs@thermofisher.com. Antibody Handbook We ve added 35,000 research reagents in 42 research areas to the Thermo Scientific Pierce Product Portfolio. Request our new 2009 Antibody Catalog! To request a copy, visit or contact your local office or distributor. Thermo Scientific Open Biosystems Custom Antibody Services Tel: or Customer Assistance abs@thermofisher.com Outside the United States, visit our website or call to locate your local branch office or distributor. These products are supplied for laboratory or manufacturing applications only. Prices listed herein were accurate at the time of printing. Visit our website for up-to-date prices.

14 Custom Antibody and Biomarker Development Service Now Part of the Thermo Scientific Pierce Protein Research Product Portfolio /09 Printed in the U.S. Contact Information Belgium and Europe, the Middle East and Africa Distributors Tel: France Tel: The Netherlands Tel: Germany Tel: United Kingdom Tel: Switzerland Tel: perbio.euromarketing@thermofisher.com United States Tel: Fax: Customer Assistance abs@thermofisher.com 2009 Thermo Fisher Scientific Inc. All rights reserved. Texas Red is a trademark of Molecular Probes, Inc. Dyomics is a trademark of Dyomics GmbH. All other trademarks belong to Thermo Fisher Scientific Inc. and its subsidiaries. Prices listed herein were accurate at the time of printing.