Contact Information Belgium and Europe, the Middle East and Africa Distributors Tel: +32 53 85 71 84 France Tel: 0 800 50 82 15 The Netherlands Tel: 076 50 31 880 Germany Tel: 0228 9125650 United Kingdom Tel: 0800 252 185 Switzerland Tel: 0800 56 31 40 Email: perbio.euromarketing@thermofisher.com www.thermo.com/perbio United States Tel: 888-412-2225 Fax: 256-704-4849 Customer Assistance E-mail: 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.
Custom Antibody and Biomarker Development Service — Now Part of the Thermo Scientific Pierce Protein Research Product Portfolio
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
•
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
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 anti-bodies. 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 car-rier 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 high-utility 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 •
For more information, or to download product instructions, visit www.thermo.com/openbiosystems
1
Antibody Services
Thermo Scientific Open Biosystems
Custom Antibody and Proteomics Services
Available from the Thermo Scientific Pierce Protein Research Products Portfolio
1601785 05/09 Printed in the U.S.
Contact Information Belgium and Europe, the Middle East and Africa Distributors Tel: +32 53 85 71 84 France Tel: 0 800 50 82 15 The Netherlands Tel: 076 50 31 880 Germany Tel: 0228 9125650 United Kingdom Tel: 0800 252 185 Switzerland Tel: 0800 56 31 40 Email: perbio.euromarketing@thermofisher.com www.thermo.com/perbio United States Tel: 888-412-2225 Fax: 256-704-4849 Customer Assistance E-mail: 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.
Custom Antibody and Biomarker Development Service — Now Part of the Thermo Scientific Pierce Protein Research Product Portfolio
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
•
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
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 anti-bodies. 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 car-rier 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 high-utility 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 •
For more information, or to download product instructions, visit www.thermo.com/openbiosystems
1
Antibody Services
Thermo Scientific Open Biosystems
Custom Antibody and Proteomics Services
Available from the Thermo Scientific Pierce Protein Research Products Portfolio
1601785 05/09 Printed in the U.S.
2
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 purifi-cation 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.
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
Polyclonal antibody packages.
Selected rabbit
polyclonal packages Options
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 • 180-200 ml serum
Peptide design synthesis and conjugation
Customer-supplied antigen Customer-supplied conjugated antigen Design, synthesis, conjuga-tion and co-injecconjuga-tion of two peptides
Protein modification specific (phosphorylation, acetylation, splice variants, etc.) with affinity-purified, negative absorbed antibody 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 • 130-150 ml serum
Peptide design synthesis and conjugation
Customer-supplied antigen Customer-supplied conjugated antigen Design, synthesis, conjuga-tion and co-injecconjuga-tion of two peptides
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 an-tibody 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.
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
Novel methodologies are used for presenting antigens in confor-mationally 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: keyhole lympet hemocyanin BSA: bovine serum albumin OVA: ovalbumin
THY: thyroglobulin
MAP: multiple antigen peptide (no conjugation necessary)
Antibody Services
For more information, or to download product instructions, visit www.thermo.com/openbiosystems
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.
Continuation protocols.
Description Service
Standard monthly
extension One boost, one bleed and maintenance of animal for one month High-dosage
extension One boost with higher dosage antigen, bleed and maintenance for one month Aggressive
extension Two boosts seven days apart at higher dosages followed by bleeding and maintenance
Holding animals One-month maintenance options with no boosting or bleeding
Terminal bleeds Exsanguination on request
Polyclonal Antibody Purification
A variety of different purification strategies are offered, depending on the needs for the antibody; however, peptide affinity chroma-tography 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.
A number of activated resins are used for immobilizing molecules, depending on the conjugation method used and project strategy.
Antibody purification options.
Description Service
Total IgG purification Ammonium sulfate precipitation Protein A/G
purification Highly specific protocol to isolate all IgG from serum or supernatant Peptide affinity
purification Immobilized peptide on affinity resin following by pH elution Affinity depletion Used to enrich for antibodies to specific
modifications (e.g., phos)
Purification Quality Assurance
The final step of the purification is to evaluate and quality- assure 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 anti- immunogen 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.
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) Immunocytochemistry (ICC) Immunofluorescence (IF) Fluorescence activated cell
sorting (FACS) Western Blotting Chromatin IP (ChIP) Immunoprecipitation (IP) Competitive inhibition
(Antagonists) ELISA
www.openbiosystems.com P: (888) 412-2225 F: (256) 319-1460 support@openbiosystems.com
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.
Project # Customer Volume (mL) Conc. (mg/mL) Total (mg) Titer Flow-through A590001 John Doe 8 0.449 3.59 5ng 256.00 AF301, AF302 A590002 John Doe 8.5 3.91 33.24 5ng 2048.00 AF303, AF304 A590003 John Doe 8 2.958 23.66 5ng 2048.00 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.
Antibody
Ser
vic
es
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 www.thermo.com/openbiosystems or
contact a project manager at abs@thermofisher.com.
Thermo Scientific Open Biosystems Custom Antibody Services
Tel: 888-412-2225 or 256-704-4848 • Customer Assistance E-mail: abs@thermofisher.com
Outside the United States, visit our website or call +1-256-704-4848 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.
www.thermo.com/openbiosystems
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 www.thermo.com/abr or contact your local office or distributor.
2
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 purifi-cation 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.
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
Polyclonal antibody packages.
Selected rabbit
polyclonal packages Options
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 • 180-200 ml serum
Peptide design synthesis and conjugation
Customer-supplied antigen Customer-supplied conjugated antigen Design, synthesis, conjuga-tion and co-injecconjuga-tion of two peptides
Protein modification specific (phosphorylation, acetylation, splice variants, etc.) with affinity-purified, negative absorbed antibody 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 • 130-150 ml serum
Peptide design synthesis and conjugation
Customer-supplied antigen Customer-supplied conjugated antigen Design, synthesis, conjuga-tion and co-injecconjuga-tion of two peptides
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 an-tibody 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.
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
Novel methodologies are used for presenting antigens in confor-mationally 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: keyhole lympet hemocyanin BSA: bovine serum albumin OVA: ovalbumin
THY: thyroglobulin
MAP: multiple antigen peptide (no conjugation necessary)
Antibody Services
For more information, or to download product instructions, visit www.thermo.com/openbiosystems
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.
Continuation protocols.
Description Service
Standard monthly
extension One boost, one bleed and maintenance of animal for one month High-dosage
extension One boost with higher dosage antigen, bleed and maintenance for one month Aggressive
extension Two boosts seven days apart at higher dosages followed by bleeding and maintenance
Holding animals One-month maintenance options with no boosting or bleeding
Terminal bleeds Exsanguination on request
Polyclonal Antibody Purification
A variety of different purification strategies are offered, depending on the needs for the antibody; however, peptide affinity chroma-tography 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.
A number of activated resins are used for immobilizing molecules, depending on the conjugation method used and project strategy.
Antibody purification options.
Description Service
Total IgG purification Ammonium sulfate precipitation Protein A/G
purification Highly specific protocol to isolate all IgG from serum or supernatant Peptide affinity
purification Immobilized peptide on affinity resin following by pH elution Affinity depletion Used to enrich for antibodies to specific
modifications (e.g., phos)
Purification Quality Assurance
The final step of the purification is to evaluate and quality- assure 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 anti- immunogen 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.
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) Immunocytochemistry (ICC) Immunofluorescence (IF) Fluorescence activated cell
sorting (FACS) Western Blotting Chromatin IP (ChIP) Immunoprecipitation (IP) Competitive inhibition
(Antagonists) ELISA
www.openbiosystems.com P: (888) 412-2225 F: (256) 319-1460 support@openbiosystems.com
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.
Project # Customer Volume (mL) Conc. (mg/mL) Total (mg) Titer Flow-through A590001 John Doe 8 0.449 3.59 5ng 256.00 AF301, AF302 A590002 John Doe 8.5 3.91 33.24 5ng 2048.00 AF303, AF304 A590003 John Doe 8 2.958 23.66 5ng 2048.00 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.
Antibody
Ser
vic
es
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 www.thermo.com/openbiosystems or
contact a project manager at abs@thermofisher.com.
Thermo Scientific Open Biosystems Custom Antibody Services
Tel: 888-412-2225 or 256-704-4848 • Customer Assistance E-mail: abs@thermofisher.com
Outside the United States, visit our website or call +1-256-704-4848 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.
www.thermo.com/openbiosystems
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 www.thermo.com/abr or contact your local office or distributor.
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
✔
✔
✔
✔
NADepleting Peptide or Control Peptide NA NA
✔
✔
Optional NAProtein Expression Optional NA NA NA NA
✔
Animal Immunization Protocol
✔
✔
✔
✔
✔
✔
ELISA Titration
✔
✔
✔
✔
✔
✔
Affinity Purification and Depletion Optional Optional
✔
✔
NA NAFusions and Cloning NA NA NA NA
✔
✔
Monoclonal Production and Purification NA NA NA NA Optional Optional
Complete Online Project Tracking
✔
✔
✔
✔
✔
✔
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.
4 For more information, or to download product instructions, visit www.thermo.com/openbiosystems
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 •
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. 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. Develop-ment 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.
Custom Monoclonal Antibodies
To order, call 888-412-2225. Outside the United States, contact your local branch office or distributor. 5
Monoclonal antibodies.
Standard Monoclonal
Packages Phase 1 and 2 Phase 3 Deliverables
5 Balb/c 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.
10 (5 BALB/c and 5 SW) Mouse Hybridoma Development
Up to 3 fusions performed. Supernatants and 5 frozen cell lines shipped.
15 (5 BALB/c and 10 SW) Mouse Hybridoma Development
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 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 proteins or protein fragments 10 (5 BALB/c and 5 SW)
Mouse Hybridoma Development
Recombinant protein fragments or peptides 15 (5 BALB/c and 10 SW)
Mouse Hybridoma Development
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 Single limiting-dilution cloning to pick out consistent producers with confirmed isotype and production. Multi-cycle complete
clonality cloning
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) •
5
6 For more information, or to download product instructions, visit www.thermo.com/openbiosystems
Custom Peptide Services
Custom Peptide Synthesis Services
We offer best-in-class custom peptide synthesis
capabilities with a wide-range of labels,
modifica-tions, scales, purities and delivery time options to
meet your research needs.
Our never-ending commit-ment 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.
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.
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 pro-cesses. 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 1999. 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 HeavyPeptideKits – Basic and AQUA Grade
for relative and absolute quantification of proteins We routinely synthesize peptides labelled with 13C and 15N
isotopes. These HeavyPeptides™ Kits are extremely useful
for quantifying your protein by mass spectroscopy. Visit
www.thermo.com/heavypeptide for more details.
To order, call 888-412-2225. Outside the United States, contact your local branch office or distributor. 7
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.
Modifications and labels available.
Acetylation of N-terminus
(Ac-NH-) Fluorescein (FITC/5-FAM) Acetyl-lysine Formic acid
Aldehyde Glycosylation (Mannose, Galactose) 6-amino hexanoic
acid (Ahx) Hydroxy proline Amidation of C-terminus
(-CONH2)
Isotopically labeled amino acids (with 2H, 13C, 15N)
Amino benzoic acid Keyhole limpet hemocyanin (KLH) Beta alanine Mercaptopropionic acid Bovine serum
albumin (BSA) Methoxy-coumarin-acetic acid (MCA)
Biotin Methyl lysine
Caproic acid Multiple antigen peptides (MAP) CBZ (Z) Myristic acid
Citrulline 3-nitro tyrosine Conjugation (BSA, KLH) Norleucine (Nle) Conjugation
(oligonucleotides) Octanoic acid Coumarin Palmitoic acid
Custom FRET peptides Phosphorylation of Ser, Thr and Tyr Cyclization via termini
or disulfidebridge Pyro glutamic acid (Pyro-E) 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
DyLight Dyes Tetramethylrhodamine (TAMRA) 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.
Quality assurance.
MADLI-TOF mass analysis LCMS Electrospray mass analysis (ES) HPLC Vapor-sealed bottle (nitrogen) AAA
Delivery
Turnaround time is key for accurately scheduling experiments. Our capabilities and throughput enable us to deliver every pep-tide 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).
Purity Scale Crude >70% >80% >90% >95% >98% 2 mg 1 1-2 1-2 1-2 1-2 2 5 mg 1 1-2 1-2 1-2 1-2 2 10 mg 1 1-2 1-2 1-2 1-2 2 15 mg 1 1-2 1-2 1-2 2-3 2-4 20 mg 1 1-2 1-2 1-2 2-3 2-4 30 mg 1-2 2 2-3 2-3 2-3 3-4 50 mg 1-2 2 2-3 2-3 3-4 4-6 100 mg 1-2 2 2-3 3-4 3-4 4-6 Ordering
Ordering is easy, Online quote forms or printable fax forms are available at: www.openbiosystems.com/Antibodies/peptides/
7
8 For more information, or to download product instructions, visit www.thermo.com/openbiosystems
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. 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 email to abs@thermofisher.com using the code BROHP07.
Amino acid Mass difference to standard AA Isotope composition
Alanine/A + 4 Da U-13C3, 98%; 15N, 98% Argenine/R + 10 Da U-13C6, 98%; 15N4,98% Isoleucine/I + 7 Da U-13C6, 98%; 15N, 98% Leucine/L + 7 Da U-13C6, 98%; 15N, 98% Lysine/K + 8 Da U-13C6, 98%; 15N2,98% Phenylalanine/F + 10 Da U-13C9, 98%; 15N, 98% Proline/P + 6 Da U-13C5, 98%; 15N, 98% Valine/V + 6 Da U-13C5, 98%; 15N, 98%
Other amino acids on request
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 power-ful tools for relative and absolute quantitation of post-transla-tional 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 • H2N-HL SASPSDNVYL R-COOH H2N COOH
H2N-HL SASP (pS)DNVYL R-COOH
H2N
COOH
H2N-HL SASPSDNVYL* R-COOH 2N-HL SASP (pS)DNVYL* R-COOH
A signature peptide is identified and sequenced
An internal standard is synthesized. A stable isotopically-labelled amino acid is incorporated during the process. The resulting product is called HeavyPeptide or HeavyPeptide AQUA
Phosphorylated Protein Native Protein phos H H2N-HL SASPSDNVYL R-COOH H2 N COOH H2N-HL SASPSDNVYL*R-COOH Heavy Protein
Figure 1. Heavy peptide quantitation.
To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch office or distributor. 9
Thermo Scientific Product Name Here
Protein Expression and Antibody
Biomarker Development
Bringing it all together – a comprehensive
platform for protein detection.
We offer a completeplatform 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 •
Custom Protein Expression
We offer bacterial and mammalian protein
expression services as part of our antibody
development platform.
Protein antigens are essential start-ing 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.Service Description Time
pLEX subcloning Subclone gene of interest in pLEX vector with c-terminal epitope tags
2 weeks
DNA prep Prepare plasmid DNA
by maxiprep 1 week
Particle
production Generate 10
6 lentiviral
particles 1 week
Transduction Transduce CHO or HEK cells Stable cells Select for stable cell lines
using puromycin 2 weeks Expression testing Overexpression testing for
positive bands 1 week Protein
confirmation By Western blot with supplied antibody or with epitope tag 1 week Scale up 10-L wave bag production
(~10-100 mg protein) 4 weeks Protein
purification Chelation and FPLC 2 weeks
Biomarker Development and Protein Expression Services
9
Figure 1. Analysis of a complete protein highlights signal peptides, structural motifs and modified residues.
Figure 2. Automated BLAST analysis compares desirable peptides across dozens of species, increasing your antibody specificity.
Antigen Design Software
Antigen Design and Presentation with
Thermo Scientific Antigen Profiler Software
The power of bioinformaticsWe 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 auto-mated 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
• www.thermo.com/openbiosystems 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 submis-sion 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.
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 •
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.
Optimal Peptide Design Algorithms
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
2
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 purifi-cation 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.
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
Polyclonal antibody packages.
Selected rabbit
polyclonal packages Options
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 • 180-200 ml serum
Peptide design synthesis and conjugation
Customer-supplied antigen Customer-supplied conjugated antigen Design, synthesis, conjuga-tion and co-injecconjuga-tion of two peptides
Protein modification specific (phosphorylation, acetylation, splice variants, etc.) with affinity-purified, negative absorbed antibody 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 • 130-150 ml serum
Peptide design synthesis and conjugation
Customer-supplied antigen Customer-supplied conjugated antigen Design, synthesis, conjuga-tion and co-injecconjuga-tion of two peptides
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 an-tibody 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.
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
Novel methodologies are used for presenting antigens in confor-mationally 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: keyhole lympet hemocyanin BSA: bovine serum albumin OVA: ovalbumin
THY: thyroglobulin
MAP: multiple antigen peptide (no conjugation necessary)
Antibody Services
For more information, or to download product instructions, visit www.thermo.com/openbiosystems
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.
Continuation protocols.
Description Service
Standard monthly
extension One boost, one bleed and maintenance of animal for one month High-dosage
extension One boost with higher dosage antigen, bleed and maintenance for one month Aggressive
extension Two boosts seven days apart at higher dosages followed by bleeding and maintenance
Holding animals One-month maintenance options with no boosting or bleeding
Terminal bleeds Exsanguination on request
Polyclonal Antibody Purification
A variety of different purification strategies are offered, depending on the needs for the antibody; however, peptide affinity chroma-tography 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.
A number of activated resins are used for immobilizing molecules, depending on the conjugation method used and project strategy.
Antibody purification options.
Description Service
Total IgG purification Ammonium sulfate precipitation Protein A/G
purification Highly specific protocol to isolate all IgG from serum or supernatant Peptide affinity
purification Immobilized peptide on affinity resin following by pH elution Affinity depletion Used to enrich for antibodies to specific
modifications (e.g., phos)
Purification Quality Assurance
The final step of the purification is to evaluate and quality- assure 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 anti- immunogen 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.
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) Immunocytochemistry (ICC) Immunofluorescence (IF) Fluorescence activated cell
sorting (FACS) Western Blotting Chromatin IP (ChIP) Immunoprecipitation (IP) Competitive inhibition
(Antagonists) ELISA
www.openbiosystems.com P: (888) 412-2225 F: (256) 319-1460 support@openbiosystems.com
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.
Project # Customer Volume (mL) Conc. (mg/mL) Total (mg) Titer Flow-through A590001 John Doe 8 0.449 3.59 5ng 256.00 AF301, AF302 A590002 John Doe 8.5 3.91 33.24 5ng 2048.00 AF303, AF304 A590003 John Doe 8 2.958 23.66 5ng 2048.00 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.
Antibody
Ser
vic
es
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 www.thermo.com/openbiosystems or
contact a project manager at abs@thermofisher.com.
Thermo Scientific Open Biosystems Custom Antibody Services
Tel: 888-412-2225 or 256-704-4848 • Customer Assistance E-mail: abs@thermofisher.com
Outside the United States, visit our website or call +1-256-704-4848 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.
www.thermo.com/openbiosystems
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 www.thermo.com/abr or contact your local office or distributor.
