ProteinTech
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Dual staining performed with Proteintech's anti-tubulin antibody 66031-1-Ig (green) and anti-SIRT6 antibody 13572-1-AP (red).
Blue counterstain: human anti-mito HMS-0200 (Immunovision). (Credit: Nancy Kedersha, Brigham and Women's Hospital, Boston).

Proteintech's Origins

"The merit of originality is not novelty; it is sincerity."

Thomas Carlyle – Scottish Philosopher

Proteintech's Origins

Proteintech Group was founded in 2002 by a group of scientists, who were actively engaged in NIH funded research at the time. One of the original founders and current CEO Dr. Jason Li was an Associate Professor of Immunology at the University of Illinois at Chicago during the company's foundation years. He says of the reasons behind Proteintech's establishment:

"At the heart of our aspiration was a company for scientists by scientists; an antibody provider that would offer researchers the advantage — not investors."

Original Products – original products

Part of Proteintech Group's early vision was to make all its own products, to the highest standards possible, and never source a single antibody from any other provider. This would offer researchers many advantages, including a way of avoiding the same antibodies sold under different brand names by different companies – an unregulated practice that has arisen within the industry1.

With one hundred percent original products Proteintech offers its customers sincerity and integrity, qualities you will discover continually throughout this brochure. You can only buy Proteintech antibodies directly from Proteintech or via one of its approved distributors — when you receive your antibody and see the Proteintech logo on the vial, know that you hold something that is truly unique.

[1] Voskuil J. Commercial antibodies and their validation [v2; ref status: indexed,http://f1000r.es/4jp] F1000Research 2014, 3:232 (doi:10.12688/f1000research.4966.2)

The Antibodies of Tomorrow…

"…The highlights of tomorrow are the
unpredictabilities of today."

Caesar Milstein, Nobel Prize Winner and Father of monoclonal technology.

Another fundamental part of the Proteintech Group vision was to produce an antibody for every single protein coded for in the human genome. Around the time of the company's inception the Human Genome Project was reaching its conclusion and the era of proteomics was dawning. Knowing that the study of proteins was key to translating the wealth of information contained within the genome, Proteintech set about providing the most vital tools for this task: antibodies — reagents without equal in the pursuit of protein research. Furthermore, the antibodies they aimed to provide were not just those relevant to the research of the day, but those of the discoveries of tomorrow. Because it is impossible to predict where science might go, and which targets may be in demand in future, comprehensive coverage of all human gene products is vital to supporting scientists.

So far Proteintech has covered up to two-thirds of the human genome with its antibody products, and reaching the last protein in the genome is still an important target today.

The Whole-Protein
Advantage

"Nature, to be commanded, must be obeyed"

Francis Bacon, English Philosopher

The benefits of whole-protein antibodies, among others, are their robustness and versatility. In addition to their capability at routine target detection – such as Western blotting for an abundant protein – they outperform peptide antibodies in exploratory science and in suboptimal conditions. For instance they show greater success in the analysis of patient samples where pathological proteins are present at endogenous levels or lower. Proteintech's anti-TDP-43 protein is a principal example of this…

Proteintech's monoclonal TDP-43 antibody (60019-2-Ig) binds to the TDP-43 protein via recognition and interaction with a specific epitope - mapped to amino residues 203-209 by Hiroshi Tsuji et al. (2012). This site is involved in the pathological aggregation of TDP-43. Note its location at the protein's surface.
Reference: H Tsuji, T Arai, F Kametani et al., Brain. 2012 Nov;135 (Pt 11):3380-91.

TDP-43

"…The species that survives […] is the one most adaptable to change."

Charles Darwin, British Naturalist and Geologist

TDP (TAR DNA binding protein)-43 is an important target in neurodegeneration research, but its story started with the Human Immunodeficiency Virus (HIV) in the mid-1990s — it was shown to bind DNA motifs of HIV origin1; however, only a dozen more publications followed in the next decade.

In 2006, findings published in the journal Science2 would ultimately change TDP-43's status from a minor research target to a mainstay of neurodegeneration research. The authors identified TDP-43 as the mystery constituent of protein inclusions in FTLD-U, one of the most common forms of frontotemporal dementia (FTD), a family of neurodegenerative diseases.

Moreover, they identified it in the overlapping disease ALS, or Lou Gehrig's disease.

Proteintech's anti-TDP-43 antibody (10782-2-AP) was used to verify mass spectrometry data through immunohistochemical and biochemical analyses of FTLD-U brain tissue samples. The group were able to visualize the inclusions in several more types of FTLD-U than first predicted, where other antibodies had failed. The whole-protein beginnings of the TDP-43 antibody enabled it to 'see' aggregated TDP-43 in more disease scenarios than the other antibodies used by the group at earlier stages of the work…

…But there's more to Proteintech's whole-protein approach than just targeting more protein…

[1] Ou SH, Wu F, Harrich D et al., (1995), Cloning and characterization of a novel cellular protein, TDP-43, that binds to human immunodeficiency virus type 1 TAR DNA sequence motifs. Journal of Virology: June 1995; 69(6): 3584–96.

[2] Neumann M, Sampathu DM, Kwong LK, et al., (2006) Ubiquitinated TDP-43 in Frontotemporal Lobar Degeneration and Amyotrophic Lateral Sclerosis. Science: October 2006; 314(5796): 130–3

Working with the Immune System

"There are no shortcuts in evolution"

Louis D. Brandeis, Former Associate Justice of the Supreme Court of the
United States

The lab employs a 102 day-long immunization protocol as standard, supplemented with booster immunizations to emulate a realistic immune response. This protocol, paired with the whole-protein approach, allows two major processes to happen in the host immune system that contribute to superior antibodies…

Selection of epitopes
by MHC class II molecules

Affinity Maturation

Up to Four Times Higher Affinity

"The greatest SMN signal with…broadest dynamic range was seen with the
combination of ER4 (buffer) and the Proteintech antibody."

D.T.Kobayashi et al., PLoS One. 2011; 6 (8): e24269

The greater recognition capabilities of Proteintech antibodies are no better demonstrated than with the example of its anti-SMN2 antibody (11708-1-AP).

Researchers at The SMA (spinal muscular atrophy) Foundation set out to develop an assay that would need just one milliliter of blood for the detection of SMN2. A tough call as SMN2 levels in SMA patients are pretty low1. Therein lay a challenge as a protein signal cannot be augmented from a small sample in the same way a trace of DNA can by PCR. Yet, with a sensitive antibody possessing high affinity, a protein can be captured and enriched enough for detection. This is the basic principle of the Sandwich ELISA procedure, and whole-protein raised antibodies are the ideal candidates for such protocols.

This is because they recognize more of the protein, in three-dimensional, native conformations.

In their efforts to develop a Sandwich ELISA assay for the detection of SMN2 in PBMCs The SMA Foundation scientists found that Proteintech's anti-SMN2 antibody (11708-1-AP) proved four times more sensitive than the nearest ranking equivalent from among its competitors1. Taking this antibody forward into their design of the assay, the SMA researchers were able to develop a reliable, quantitative and rapid method for the measurement of SMN2 in SMA patient PBMCs. In addition, the resulting assay was also able to detect SMN in several tissues of mouse origin, including a mouse model of severe SMA1.

The Whole Protein approach is not the only measure Proteintech takes to ensure the quality of its antibodies. Read on to find out more about its production and validation procedures…

[1] Kobayashi DT, Olson RJ, Sly L et al. (2011) Utility of survival motor neuron ELISA for spinal muscular atrophy clinical and preclinical analyses. PLoS One: August 2011; 6(8):e24269.

Validation

"As a scientist, you're not supposed to make decisions without the data."

Francis Sellers Collins, American physician-geneticist and Director of the
National Institutes of Health

Study the schematic, which shows our validation hierarchy. You'll notice that we don't sell any antibodies that do not have a valid Western blot (WB). In exceptional circumstances we approve products for sale that have

been reviewed by an independent medical pathologist for use in immunohistochemistry (IHC) without WB data. All validation experiments are performed using unmodified samples and endogenous levels of proteins.

Open Access Data

The scientific community thrives when data is shared freely. Without data sharing, experiments cannot be peer-reviewed, and scientists cannot validate existing findings by repeating or improving experiments. At Proteintech we believe data sharing does not just fall within the sphere of the research community – we know our validation data can contribute something to science too. Therefore, we have an open access data policy to assist you with your initial antibody purchase and to facilitate your experimental

design. All of Proteintech's validation results are accessible on its website, and the scientist responsible for those data can be reached via Proteintech's technical help channels at any time.
Furthermore, we make available any data your research peers have shared with us, with permission, so you can see how an antibody has performed in the hands of others. We encourage any feedback, positive and negative, from all of our customers.

Continuing Commitment and Support

"Commitment is an act, not a word."

Jean-Paul Satre, French Philosopher

The pursuit of our scientific ideals, our extensive validation procedures and the previous success stories of Proteintech antibody users, all attest to a scientific company that puts its customers and their research first.

Recently, Proteintech has pledged to undertake siRNA knockdown validation of most of its antibodies by the end of 2015. The specificities of over 550 of its antibodies have already been confirmed by customers using siRNA knockdown, and published in over 650 journal papers. In adding its own siRNA data to those previously available in the literature, Proteintech's aim is to make a gold standard of antibody validation more accessible to its customers. In doing so, Proteintech will be setting a new benchmark for antibody validation – one that crosses the boundary between provider and user, and absolves the question of with whom the responsibility of antibody validation should fall.

Proteintech is now committed to realizing this ambition, in addition to continuing its support of scientists beyond an antibody's arrival at the lab bench. At Proteintech we understand that the success stories of its antibodies only really start once they are in your hands, so we will go that extra mile to ensure that, in a discipline with so many variables, you have a trusted source of consistent, quality antibodies.

Defining Success

10,000 success stories and counting

How do you define success? It is a question every company must explore from the outset. It characterizes a business' culture and approach to its customers. Essentially, it is a question that provides the framework for a company's operation and ethics. Success for Proteintech and its team isn't having 12,000 products in its catalog, nor is it seeing its sales increase. What really matters to Proteintech and the team here is the success of its antibodies in the hands of scientists – the people who make the discoveries that change and explain the world around us. They need well-validated antibodies they can really rely on, reagents they can trust to reveal true data. This is the axis about which Proteintech operates and prospers, valuing every item of peer-reviewed research published by its customers. It is the rapidly growing number of research papers featuring its antibodies that Proteintech counts as real, tangible success. With 10,000 publications and counting the numbers speak for themselves…


So what will your success story be?

Search the ProteinTech catalog to begin…
Indirect Elisa

Determination of Antibody titer

Affinity Purification
Western Blotting

Using whole, unmodified cell or tissue lysates

Immunoprecipitation
Flow Cytometry
siRNA Knockdown
Immunohistrochemistry
Immunofluorescence
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Proteintech's polyclonal (10782-2-AP) and monoclonal (60019-2-Ig) anti-TDP-43 antibodies stain FTLD-U autopsy specimens (viewed at 40x). Staining shows dystrophic neurites in accordance with the literature. (Credit: Linda K. Kwong, Center for Neurodegenerative Disease Research & Institute on Aging, University of Pennsylvania.)

Indirect ELISA Testing

All antibodies, whether polyclonal or monoclonal, are screened for recognition of the antigen and their relative abundance by indirect ELISA testing.

In this procedure, a microwell plate is coated with the target antigen in replicates of differing concentrations and used to capture any specific antibody from sera or hybridoma supernatants. Captured antibody is then detected indirectly using enzyme-coupled, species-specific secondary antibody. This experiment reveals antibody titer i.e. the measurement of how much antibody is present in a batch of serum that recognizes a particular epitope, expressed as the inverse of the greatest dilution that yields a positive result.

ELISA testing enables us to screen hundreds of monoclonal subclones or decide which batch of polyclonal antisera to take forward into purification and further validation testing.

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Affinity Purification

Following the isolation of host anti-serum and titration by indirect ELISA, the next step we take to ensure the quality of our polyclonal antibodies is to carry out affinity purification. This is performed using antigen-coupled sepharose, meaning only those antibodies specific to the immunogen are purified from the serum.

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Western Blotting

A successful Western blot experiment is an essential validation requirement: we do not approve an antibody for sale until we have supporting Western blot data. All our blotting experiments are performed strictly using either endogenous tissue or unmodified cell line lysates. If a product fails to generate a clean, specific signal it is rejected and redeveloped. If no signal is seen and the endogenous protein levels are low, we review the product with immunoprecipitation experiments. If the target is expressed in difficult-to-obtain tissue, we hand the antibodies over to medical pathologists for their assessment. In certain circumstances these pathologists approve targets with supporting immunohistochemistry data.

If successful at the Western blotting stage, an antibody is released for sale under our guarantee, but validation does not stop there…

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Immunoprecipitation

With immunoprecipitation, it's a similar story to immunohistochemistry: whole-protein antigens produce antibodies with superior sensitivity. Plus resulting antibodies are optimized for surface epitopes as well as recognizing 3-D conformations, meaning they will form superior immune complexes with native proteins in immunoprecipitation assays.

Proteintech's immunoprecipitation department is currently focused on capturing and detecting all those protein targets with low endogenous protein expression – especially those with levels below the current threshold of Western blot detection (some 30% of the genome!) In 40% of validation cases where Western blotting experiments had failed previously, IP enrichment with a corresponding Proteintech antibody has ensured successful Western blotting.

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Flow Cytometry

An increasing number of Proteintech's customers now require antibodies that are approved for use in flow cytometry. In response, Proteintech has added this application to its validation workflow.

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siRNA Knockdown

Proteintech will undertake siRNA knockdown validation of most of its antibodies by the end of 2015. This will further demonstrate the specificity of its products and help to identify any candidates in need of redevelopment. The first datasets from this project are now being uploaded to the website. Read on to find about Proteintech's motivations for adding siRNA knockdown to its validation repertoire.

siRNA Knockdown of AKT1 detected with Proteintech's anti-AKT1 antibody (10176-2-AP, red). Three transfection periods: 48, 72 and 96 hours (L-R) were assessed for siRNA transfection efficiency. Cultured cells were incubated with mock siRNA, 3nM control siRNA, or 5, 10 or 20nm siRNA targeting AKT1 mRNA (L-R) for the three time periods. A loading control antibody (green) was also used to check for even sample loading at SDS-PAGE. (A molecular weight marker can be seen in the far-left lane.) Credit: Dr. Eva Martinez-Balibrea.

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Immunohistochemistry

The nature of whole-protein raised antibodies makes them ideal candidates for application in immunohistochemistry (IHC). If epitopes become damaged or hidden during IHC sample preparation, whole-protein raised polyclonal antibodies can compensate. The IHC stage of the validation protocol puts this theory to the test. Around 60% of Proteintech antibodies have been successful in IHC, with further products undergoing testing.

Even Proteintech's monoclonal antibodies have a good IHC success rate, an application for which finding suitable monoclonals can be challenging: 49% of its monoclonal antibodies show positive staining patterns in IHC experiments, in a range of different tissues from a variety of species.

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Immunofluorescence

Proteintech also screens its antibodies against target proteins and their specific cellular locations in immunofluorescence. This supports specific Western blot data by establishing whether the localization of the target protein is also in agreement with the literature.

Image Credit: Nancy Kedersha of Brigham and Women's Hospital, Harvard University

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[Left image:]
The immunogen is taken up by host antigen presenting cells,

[Right image:]
where it undergoes lysosomatic processing before presentation by MHC class II molecules to T Cells…

Many of the peptide epitopes produced by lysosomal processing would go on to generate a suboptimal immune response were the process of antigen presentation to be non-selective. However the binding-groove of the MHC class II molecule is adapted to select the epitopes with the best chances of eliciting a response. The identity of these optimal epitopes is difficult to predict using current algorithms for peptide antigen design.

Another issue of using artificially designed peptide antigens lies in the interaction between the T-cell and the MHC class II molecule. MHC class II molecules are crucial to T-cell selection during T cell maturation. Since the T cell population is "trained" for epitope presentation by the host's individual MHC II molecules only the subset of epitopes "preferred" by the MHC molecule will generate the strongest T-cell response.

[Top images]
The core binding motif of MHC II is approximately nine amino acids long. Unlike the closed binding groove of the MHC I molecule the ends of the MHC II binding groove are open.

Consequently, MHC class II can accommodate much longer peptides – possibly even whole proteins.
All this means:
• Selection of the best natural epitope(s).
• Greater recognition of 3D antigen motifs.

Though an initial population of B-cells produces antibodies with sufficiently high enough affinity for activation by T-cells, their affinity is not yet high enough to facilitate an effective immune response…

The means by which antibody affinity is enhanced is a microevolutionary process called affinity maturation, which happens in the peripheral immune system – mainly in lymph nodes like the one shown in this histopathology slide.

In the lymph node a process called somatic hypermutation (SHM) happens in a population of B-cells known as centrocytes. SHM is a process whereby the variable region of B-cell DNA encoding the antibody antigen binding domain, undergoes many point mutations introduced by a molecule called AID.

SHM increases (or decreases) the affinity of an individual centrocyte's antibody population. The modified antibody population is then displayed on surface of the centrocyte. These antibody populations with altered antigen affinities then compete for antigen binding and subsequent activation of their specific centrocyte by T-cells and, ultimately, their survival.

Many different centrocyte clones are produced in the process of competitive selection. Lower affinity centrocytes, which fail to capture antigen undergo apoptosis. Only those centrocytes with the highest affinity antibodies survive.

They will continue to mutate and divide, again the range of affinities provides the basis for selection by competitive binding. As cells that fail to bind activating T-cell partners undergo apoptosis, affinity is increased with each generation.

Many different centrocyte clones are produced in the process of competitive selection. Lower affinity centrocytes, which fail to capture antigen undergo apoptosis. Only those centrocytes with the highest affinity antibodies survive.

They will continue to mutate and divide, again the range of affinities provides the basis for selection by competitive binding. As cells that fail to bind activating T-cell partners undergo apoptosis, affinity is increased with each generation.

Many different centrocyte clones are produced in the process of competitive selection. Lower affinity centrocytes, which fail to capture antigen undergo apoptosis. Only those centrocytes with the highest affinity antibodies survive.

Many different centrocyte clones are produced in the process of competitive selection. Lower affinity centrocytes, which fail to capture antigen undergo apoptosis. Only those centrocytes with the highest affinity antibodies survive.

They will continue to mutate and divide, again the range of affinities provides the basis for selection by competitive binding. As cells that fail to bind activating T-cell partners undergo apoptosis, affinity is increased with each generation.

Many different centrocyte clones are produced in the process of competitive selection. Lower affinity centrocytes, which fail to capture antigen undergo apoptosis. Only those centrocytes with the highest affinity antibodies survive.

They will continue to mutate and divide, again the range of affinities provides the basis for selection by competitive binding. As cells that fail to bind activating T-cell partners undergo apoptosis, affinity is increased with each generation.

This is why Proteintech's production protocol includes a minimum of 102 days for affinity maturation.

And why booster immunizations are given at day 28. This aids the mounting of a secondary immune response and produces a greater antibody yield with higher affinity for the target.

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