Glycobiology Solutions for Your Research and Development Needs An Esoteric Discipline?

Glycans are nearly ubiquitous in eukaryotes and are essential for many cellular activities. However, for non-specialists, glycobiology has the reputation of being an esoteric discipline. It is among the least understood biological disciplines for several reasons including conceptual complexity and the lack of useful reagents.

Our Mission Bio-Techne scientists have advanced the Glycobiology field with many peer- reviewed scientific publications. The outcome of our research and development enterprise is a broad array of solutions for your glycobiology research and development needs. Our mission is to provide useful solutions that will increase the accessibility of glycobiology research to specialists and non-specialists alike.

Table of Contents

What is Glycobiology?...... 3 What is Glycosylation?...... 3 Why is Glycosylation Important?...... 3 How Can We Help You?...... 3 Quickly Screen Your Antibody or Biologic Using the Simple Western™ Platform...... 4 Use the iCE Platform as Part of Your mAb Fragmentation Analysis Workflow...... 4 Visualize Terminal Glycans Using Fluorescence or SDS-PAGE...... 5 Spatially Map Glycoprotein and Proteoglycan Biomarker Expression with RNAscope® In Situ Hybridization Assays...... 5 Use Glycoenzymes to Understand Glycan Structure and Function ...... 6 Detect Glycoproteins and Proteoglycans Using Our Validated Antibodies...... 6 Measure Glycoenzyme Activity with Our Glycobiology Assay Kits...... 7 Use High-Quality Small Molecules and Peptides for Glycobiology Research...... 7 Contact Custom Services for Bulk or Custom Analytic Solutions...... 7

bio-techne.com/glycobiology 2 What is Glycobiology Common Glycans

Glycobiology is defined as “the study of the structure, biosynthesis, biology, and evolution of D-Glucose (Glc) saccharides (also called carbohydrates, sugar chains or glycans) that are widely distributed in nature and of the proteins that recognize them” (1).

D-Mannose (Man) What is Glycosylation?

Glycosylation is a dynamic post-translational modification that produces structural differences that fine tune the functions of proteins, lipids and other glycans. Glycans are some of the D-Galactose (Gal) most structurally varied molecules in nature. This extensive diversity is due to both the large number of existing mono-saccharides and the different glycosidic linkages that can form between individual monosaccharide units. Unlike proteins and nucleic acids, glycan synthesis is not template based. Instead, glycosylation is primarily determined by the availability of N-Acetyl-D-glucosamine (GlcNAc) specific substrates and the presence of enzymes that regulate the addition and removal of glycan moieties. The increasing availability of research reagents, including tools for glycan visualization as well as regulatory enzymes such as glycosyltransferases, glycosidases, sulfotransferases and sulfatases offers a unique opportunity to advance the field of glycobiology. N-Acetyl-D-glalactosamine (GalNAc)

Why is Glycosylation Important? Fucose (Fuc)

1. Glycans play a role in communication between cells and their external environment. 2. Glycosylation is important for protein folding and quality control. Sialic Acid (Sia) 3. Glycans play an important role in the development of multicellular organisms. 4. Glycosylation is an important determinant of the efficacy of biologics such as therapeutic antibodies.

How Can We Help You?

I want to quickly assess the glycosylation of my antibody or biologic I want to alter the glycosylation of my target of interest Our Simple Western and iCE platforms help you quickly screen We also have the broadest selection of glycoenzymes. biologics prior to initiating a more involved assessment. See page 6 See Page 4 I want to assess the enzymatic activity of my glycoenzyme I want to visualize terminal glycans on cells or proteins We have kits to assay glycosyltransferase, sialyltransferase and We offer the most specific technology on the market for visualizing sulfotransferase enzymatic activity. terminal glycans on cells and proteins. See Page 7 See Page 5 I want to selectively target glycoproteins I want to know which glycoprotein or proteoglycan is expressed in We offer small molecules and peptides for targeting glycoproteins. my cell or tissue See Page 7. We offer the largest selection of glycoprotein and proteoglycan antibodies or RNA in situ hybridization probes available. I want help with my custom glycan project See Pages 5 and 6 Start a conversation with our custom services department. See page 7

1. Varki A, Cummings RD, Esko JD, et al., editors. Essentials of Glycobiology [Internet]. 3rd edition. Cold Spring Harbor (NY): Cold Spring Harbor Laboratory Press; 2015-2017. Available from: https://www.ncbi.nlm.nih.gov/books/NBK310274

bio-techne.com/glycobiology 3 Quickly Screen Your Antibody or Biologic Using the Simple Western™ Platform

Our Simple Western capillary electrophoresis platform performs automated glycan analysis using workflows that are simple and straightforward. Use these assays as a quick screening method to prior to initiating more traditional and labor-intensive methods for glycan analysis. This assay uses glycan binding proteins (Lectins) in a manner similar to antibodies to probe for glycans. Don’t want to make a capital purchase? Contact us for custom target validation and optimization services using the Simple Western platform.

18000 - PNGase F PNGase F Con A 16000

kDa TP Con A WGA TP Con A WGA 14000 Deglycosylated 230 Untreated 180 12000 116 10000 66 8000 Heavy 6000 chain 40 PNGase F 4000

Chemiluminescence Light 2000 chain 0 12 8.4 8.6 8.8 9 9.2 9.4 pl

Use Peggy Sue for charge-based lectin blots. Charge based Use Jess or Wes for size-based lectin blots. Glycosylation of capillary electrophoresis was used to assess the the NIST mAB heavy chain was visualized via size based glycosylation of the reference monoclonal antibody (mAb), capillary electrophoresis using the glycan binding proteins NIST RM8671. A large peak, representing the binding of Con A and Wheat Germ Agglutinin (WGA). No glycosylation the lectin Concanavalin A (Con A) to the glycan Mannose was observed in samples that were deglycosylated using was absent when NIST was deglycosylated. PNGase F.

Use the iCE Platform as Part of Your mAb Fragmentation Analysis Workflow

Monoclonal antibody fragmentation is an important quality control step in the manufacturing of mAbs. During this process, nonglycosylated heavy chain (NGHC), heavy chain (HC) and light chain (LC) are resolved in order to assess mAb lot-to-lot consistency. Maurice automates capillary electrophoreses to expedite this step in the manufacturing process. Maurice consistently resolves NGHC from HC with more sensitivity than traditional SDS-PAGE chip-based electrophoretic separation systems.

0.0040 240.00 0.0038 0.0036 Internal 220.00 0.0034 Standard 220.00 0.0032 0.0030 180.00 0.0028 150.00 0.0026 0.0024 140.00 0.0022 120.00 0.0020 0.0018 RFU 100.00 0.0016 80.00 0.0012 Spike 1 Absorbance 0.0012 60.00 Spike 2 0.0010 Spike 2 NGHC 40.00 0.0008 Spike 1 HL Thio NGHC HL Thio 0.0006 20.00 0.0004 0.0002 0.00 LC HC 0.0000 LC HC -20.00 -0.0002 0.80 1.00 1.20 1.40 1.60 1.80 0.280 0.320 0.360 0.400 0.440 RMT Minutes Use Maurice for mAb fragmentation Analysis. Under reduced sample conditions, the dominant light chain (LC) and HC peaks plus the major LC impurities or spikes (rhTRIP-4, spike 1; reMAP, spike 2) are detected by both Maurice (left) and LabChip® (right), but notably better resolution between the LC peak and two spikes were observed with Maurice. On Maurice, the LOD was 1.5 µg/mL and 1.9 µg/mL for Spike 1 and Spike 2, respectively, whereas the LOQ was 11.5 µg/mL (Spike 1) and 9.6 µg/mL (Spike 2). Using LabChip®, the LOD and LOQ could only be determined for Spike 1 (0.5 µg/mL and 1.7 µg/mL, respectively) due to poor resolution between Spike 2 and the LC peak.

bio-techne.com/glycobiology 4 Visualize Terminal Glycans Using Fluorescence or SDS-PAGE

Selective and specific labeling of terminal glycans has historically been a challenge due to the lack of high-affinity reagents. Our technology takes advantage of the selective action of glycosyltransferases on substrate glycans to provide the most specific glycan visualization assay on the market. Our Terminal Glycan Assay allows you to visualize glycan subcellular localization as well as protein glycosylation using standard fluorescence or SDS-PAGE technologies.

ST3GAL1 ST3GAL1 N MW

β1-4

UDP N Fetuin S/T B4GalT1Y285L S/T Desialated Fetuin

GlcNAc GlcNAc AI Protein Staining

Sialic Acid Labeling

Streptavidin-HRP detection

The glycoenzyme Human B3GALNT2 or the mutant enzyme, nylu The top gel shows total protein staining. Bovine Fetuin (left side) nylu B4GalT1Y285L, can transfer GalNac to O-GlcNAc residues. UDP- and desialated fetuin (right side) were incubated with increasing Azido GalNAc is the donor substrate in this reaction. In this amounts of recombinant enzyme Human ST3GAL1 (5,25,50, example, azido-GalNac is installed on the acceptor GlcNAc at 250 ng) and 0.5 nmol of the donor substrate CMP-Azido- position β1-4 in HUVEC cells. This reaction was visualized using Sialic Acid. The reactions were conjugated with Biotinylated Alexa-Fluor 555. Fluorescence was observed in the nucleus. Alkyne, separated by SDS Page, transferred to nitrocellulose and DAPI was used to stain nuclei. Terminal O-GlcNAc is localized to detected with Streptavidin-HRP via chemiluminescence. the nucleus and cytoplasm in HUVEC cells under these Incorporation of Azido Sialic Acid is only detected on desialated conditions, as visualized by azido-GalNac fluorescence fetuin (right side of bottom gel) indicating that ST3GAL1 sialylates fetuin only after is desialylated.

Spatially Map Glycoprotein and Proteoglycan Biomarker Expression with RNAscope® In Situ Hybridization Assays

Glycoproteins and proteoglycans are commonly identified as biomarkers in the context of cancer and other diseases. Our ready to use RNAscope® assays, based on the proprietary RNAscope® in situ hybridization (ISH) technology, allow you to visualize the expression of any target glycoprotein or proteoglycan of interest morphologically within the tissue context. These assays provide options for chromogenic or fluorescent detection and allow you to simultaneously interrogate up to 2 or 3 targets, respectively. Our RNAscope® ISH assays can also be used with the automated tissue stainers Leica BOND RX System by Leica Biosystems and Ventana DISCOVERY ULTRA system by Roche Tissue Diagnostics.

Mucin 1 (MUC1) is an o-glycosylated oncoprotein that has been correlated with cancer progression. Here the RNAscope® assay was used to visualize MUC1 gene expression in human breast cancer tissue.

bio-techne.com/glycobiology 5 Use Glycoenzymes to Understand Glycan Structure and Function

Oligosaccharide or glycan tree structure is a critical determinant of glycan function. Since the structure is determined by enzymatic activity, glycoenzymes represent a unique tool for gaining insight into glycan function. We have over 40 years of experience in developing high-quality enzymes and we provide the largest selection (4X the competition!) of highly-purified, active mammalian and bacterial recombinant glycoenzymes! • Carbohydrate Kinases • GalNAc Transferases • Hexosaminidases • Carbohydrate Sulfotransferases • GlcNAc Transferases • Neuraminidases • Fucosidases • Glucosidases • Pentosyltransferases • Fucosyltransferases • Glucosyltransferases • Protein Sulfotransferases • Galactosidase • Glucuronosyltransferases • Sialyltransferases • Galactosyl Transferases • Glycosidases • Sulfatases

OH = HO O HO = H2O OH Sulfatases OH = = Glycosidases or N or N HO O = = HO Sulfotransferases OH NH NH Glycosyltransferases 2 2 OH O O N O N O O N N HO O O S O P O O O HO O O O O N P N NH NH O O O N O N OH O O P O P O N O O P O P O O O O N OH OH OH OH 3′,5′-ADP PAPS O OH O OH O P O O P O OH OH OH OH O O UDP-glucose UDP

Glycosyltransferases catalyze the transfer of a mono- Sulfotransferases catalyze the transfer of a sulfate group to nylu saccharide moiety from a nucleotide sugar or lipid a hydroxyl or amine moiety on various molecules, including phosphosugar to acceptors such as glycans, proteins or carbohydrates and proteins. In contrast, sulfatases lipids. Glycosidases hydrolyze glycosidic linkages to release catalyze the hydrolysis of O- and N-sulfate esters from monosaccharides or oligosaccharides. these molecules.

Detect Glycoproteins and Proteoglycans Using our Validated Antibodies

We offer over 2,000 glycoprotein and proteoglycan antibodies. Count on our highly-validated antibodies for reproducible results. Our antibodies fit into your workflow and give you the right answers the first time and every time.

nylu Klotho was detected in perfusion fixed frozen sections of mouse Novuslu kidney using 1.7 µg/mL Goat Anti-Mouse Klotho Antigen Affinity- purified Polyclonal Antibody overnight at 4 °C. Tissue was stained with the Anti-Goat HRP-DAB Cell & Tissue Staining Kit and counterstained with hematoxylin (blue).

bio-techne.com/glycobiology 6 Measure Glycoenzyme Activity with Our Glycobiology Assay Kits

Assaying glycosyltransferase activity can be challenging. Historically these assays have involved the use of radiolabeled glycans. We offer inexpensive, non-radioactive assays for analyzing the activity of glycoenzymes including glycosyltransferases, sialyltransferases, and sulfotransferases. Use this 96-well plate assay to measure glycoenzyme kinetics.

100

80

60 The ability of Recombinant Human b-1,4-Galactosyltransferase nylu 1 (B4GalT1) to transfer galactose from UDP-galactose to 40 N-acetyl-alpha-D-glucosamine was measured using the Activity (pmol/min) Activity Glycosyltransferase Activity Kit. The activity was plotted against 20 SA 1806 pmol/min/µg enzyme input, and the specific activity of B4GalT1 was R2 0.9967 determined to be 1806 pmol/min/µg.

0 0 0.01 0.02 0.03 0.04 0.05 0.06 B4GalT1 (µg)

High Quality Small Molecules and Peptides for Glycobiology Research

Small molecules and peptides are the among the most effective tools for understanding mechanism of action. As your trusted supplier of innovative and high-performance life science reagents, we offer a selection of glycoprotein inhibitors and activators, including those for integrins. We are committed to providing compounds with extremely high purity.

Ac-Pro-His-Ser-Cys-Asn-NH2

ocrilu ATN 161 is a α5β1 integrin receptor antagonist, which reduces MLL tumor growth in rats and decreases tumor volume of human MDA-MB-231 cell xenografts in mice.

Contact Custom Services for Bulk or Custom Analytic Solutions

Bio -Techne is pleased to partner with you for custom and bulk requests. Contact us for custom glycan analytic testing services. We also offer a broad range of custom solutions including: • Antibody Services and Panels • Custom cDNA Cloning

• Bioactivity Testing Services • Custom Compound Libraries

• Biomarker Testing Services • ELISA Services

• Chemistry services • Luminex Services

• Custom Cell Culture Services • Protein Services

bio-techne.com/glycobiology 7 Bio-Techne and its family of world class brands are a trusted member of the glycobiology community. We are committed to glycobiology research and development by assuring the quality of our scientific publications, products, instruments and services.

THE MOST SELECTIVE TECHNOLOGY >1000 GLYCOPROTEIN ANTIBODIES

>40 YEARS OF EXPERIENCE >400 PROTEOGLYCAN ANTIBODIES

>200 CONTROL >35 GLYCOSYLTRANSFERASES LYSATES

SINGLE-COPY RNA DETECTION >20 INTEGRIN INHIBITORS X20

Ac-Pro-His-Ser-Cys-Asn-NH2

>300 GLYCOBIOLOGY PROBES AND CONTROLS >30 YEARS OF EXPERIENCE

NEW PROBES DESIGNED <1 DAY WORKFLOWS AND IN 2 WEEKS TIME TO RESULT

AUTOMATED ASSAYS 3 ORDERS OF MAGNITUDE DYNAMIC RANGE FOR SIMPLE WESTERN

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