Cytokine and Protein User’s Guide Book

RnDSy-lu-2945

2

Table of ContentsChapter 1 - Cell Culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Maintenance of Dependent Cell Lines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Protease Inhibitors for Cell Lysates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Cell Proliferation and Viability Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Development of Serum-free Media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Cell Adhesion Matrix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Chapter 2 - Lineage Differentiation/Activation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Induce or Block Differentiation and Activation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

MonitorDifferentiationEfficiency . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Chapter 3 - Protein Interaction Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Chapter 4 - Protein Processing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Signal Peptide Cleavage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Propeptide Removal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Protein Activation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Protein Degradation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Chapter 5 - Antibodies and Immunoassays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Immunogen Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Vaccine Development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Immunoassay Development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Antibody Characterization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

AntibodyPurification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Chapter 6 - Protein Characterization. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Protein Biological Activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Chapter 7 - Tissue Regeneration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

ArtificialTissueFormation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Chapter 8 - Bioprocessing Enzymes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Chapter 9 - In vivo Animal and Human Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Chapter 10 - General Resources . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

ProteinSpecificActivity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Protein Detection Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Freeze/ThawandAliquotting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Cross-Functionality . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Post-TranslationalModifications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

PredictionandComputationTools.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

3-4

4

4

4

4

4

5-6

5

5

7

7-8

7

7

8

8

9

9

9

9

9

9

10

10

11

11

12

13

14-15

14

14

14

15

15

15

3

Chapter 1 - Cell CultureMaintenance of Dependent Cell Lines

Manycelllinesrequiretheadditionofspecificgrowthfactors to their culture media evenifthemediacontainsserum.Thistablelistscelllinesandthegrowthfactorsupplement(s)theyneedtogrowattheirbest.

Chapter 1 Chapter 2 Chapter 3 Chapter 4 Chapter 5 Chapter 6 Chapter 7 Chapter 8 Chapter 9 Chapter 10

Cell Line Protein Supplements

2E8 IL-17

32 DB A5 IL-3

4mBr5 EGF

7TD1 IL-6

AML12 Insulin | Transferrin

AML-193 GM-CSF | Insulin | Transferrin

ANBL-6 IL-6

AS-E2 EPO

B9 IL-6

BaF/3 IL-3

BT-549 Insulin

CT230-ULBP1 IL-3

CT231-ULBP3 IL-3

CT29-ULBP2 IL-3

CT62-RL4 IL-3

CT7-MICA IL-3

CTLL-2 IL-2

D10N4M IL-2 | IL-1 beta

DA3 IL-3

Dendritic cells IL-4 | GM-CSF

DS-1 IL-6

EBT-8 IL-2

ELF-153 GM-CSF

EOC2 M-CSF

F-36P GM-CSF | IL-3

FBHE FGFbasic

FDC-p1 IL-3

FDCP-2 IL-3

FKH-1 G-CSF

FLAM-76 IL-6

GF-D8 GM-CSF

GGE HGF

GM/SO GM-CSF

HANK1 IL-2

HEK001 EGF

HK-2 EGF

HML GM-CSF

HPAC EGF | Insulin | Transferrin

HSM-2 IL-6

Cell Line Protein Supplements

HT-2 IL-2

HT2cloneA5E IL-2

HU-3 GM-CSF | IL-3 | Tpo

HuT102 IL-2

HUT-78 IL-2

HUVEC VEGF

JAWS II GM-CSF

JJN-2 IL-6

KHYG-1 IL-2

Kit225 IL-2

KPMM2 IL-6

M07e GM-CSF

MB-02 GM-CSF

MC/9-2 SCF | IL-4

MCF-10A Insulin | EGF

MCF-7 Insulin

MDS92 IL-3

M-MOK GM-CSF

MNFS-60 M-CSF

mNSC FGFbasic

Mo7e IL-3

MUTZ-2 SCF

MUTZ-3 GM-CSF | IL-3

MV 411 GM-CSF | Insulin | Transferrin

My-La IL-2

N1186 IL-2

N1186.94 IL-2

NCI-H345 Insulin | Transferrin

NFS-60 IL-3

NHDF-neo Insulin | FGFbasic

NK-92 IL-2

NKL IL-2

NK-YS IL-2

NSC002 EGF | FGFbasic

OCI/AML1 G-CSF

OCI/AML5 GM-CSF

OHN-GM GM-CSF

Cell Line Protein Supplements

OIH-1 G-CSF | GM-CSF

OVCAR-3 Insulin

PCM6 IL-6

PT-18 IL-3

RSC FGFbasic

Se-Ax IL-2

SKM-1 GM-CSF

SKNO-1 GM-CSF

Splenocytes IL-2SUP-HD1 IGF-I

T11 IL-11

T1165.85.2.1 IL-6

T47D Insulin

TALL-101 GM-CSF

TALL-103/2 IL-2

TALL-104 IL-2

TALL-107 IL-2

TALL-4 IL-2

T-CellBlasts IL-2

Tera-2 LIF

TF-1 GM-CSF

TF-72 IL-3

TMD2 IL-3

TN922 GM-CSF

Tramp-C1 Insulin

TSU621MT G-CSF

UCSD/AML1 GM-CSF

UG3 GM-CSF | IL-3

UT-7 GM-CSF | IL-3 | Tpo

WEHI-3 IL-3

XG-1 IL-6

XG-2 IL-6

XG-3P IL-6

XG-4 IL-6

XG-5 IL-6

XG-6 IL-6

XG-7 IL-6

YNH-1 GM-CSF

4

Protease Inhibitors for Cell Lysates

Most intracellular proteases are found in lysosomes, sequesteredfromothercellularcomponents.Theseparationis important so they don’t destroy proteins that are vital to the cell’s function. When you make cell lysates, though, you disrupt lysosome structure and give the proteases access to everythinginthelysate.Ifyouwanttopreventdegradationofyour protein, add a cocktail of protease inhibitors to block a widerangeofproteolyticactivities.

Cell Proliferation and Viability Reagents

Confirmthatthegrowthfactorsyouprovidetoyourcell culturesareworking.Monitortheproliferation and viability of your cultures.

Development of Serum-free Media

Ifyou’dliketogetawayfromusingseruminyourcellcultures,youmaybeabletoidentifygrowthfactorstouseasserumsubstitutes. Using serum-free media reduces the number of unknownfactorsinyourcellcultureandallowsyoutointerpretyourexperimentalresultswithmoreconfidence.

Sometimes you need to use serum, but you still have choices. Therearemultiplegradesoffetal bovine serum including for specializedcultureofparticularcelltypes.Thinkofitas choosing the optimum grade of a protein cocktail!

Cell Adhesion Matrix

Manycelltypesrequireasubstratetogrowwellinculture. Youcanbuildasupportivematrixwithextracellular matrix molecules and proteoglycans.

Alternatively, you can use basement membrane extract and ECMpreparationstoprovidemorecomplex(butundefined)matrix for your cultures.

Cells express a range of adhesion molecules that mediate their attachmenttotheECMandtoothercells.Youcanusesolubleforms of these proteins or adhesion molecule modulators to blockspecificinteractions.

Cell Culture Protocols

Neural Cell Culture Protocols

Neural Cell Culturing Guide

Organoid and 3-D Cell Culture Protocols

Stem Cell Culture Protocols

Cell Culture Resources

Immune Cell Culture

Neural Cell Culture

Organoid and 3-D Cell Culture

Stem Cell Culture

Molarity Calculator

Thiscalculator makes it a breeze to figureouthowmuchofaproteinyouneed to reach the desired concentration in your stock solution or media.

Chapter 1 Chapter 2 Chapter 3 Chapter 4 Chapter 5 Chapter 6 Chapter 7 Chapter 8 Chapter 9 Chapter 10

5

Chapter 2 - Lineage Differentiation/ActivationYoucaninducecellstodifferentiateintoasubsetorlineagewithadefinedcombinationofcytokinesandsmallmolecules.Thesemoleculestriggertheactivationorinhibitionofmastertranscriptionfactorsandtheexpressionofkeyproteinswhichcanbemonitoredtoconfirmthattheintendeddifferentiationhas occurred.

Thistableshowshowspecificcellsubsetscanbeinducedoractivated,thekeytranscriptionfactorsinvolved,andtheexpressionofsignaturemoleculestoconfirmtheinducedphenotype.

Induce or Block Differentiation and Activation

Cytokinesandgrowthfactors to activate cell surface receptors

Small molecules to modulate receptors and signal transduction

Blocking/neutralizingantibodiestointerferewithcytokine function

Monitor Differentiation Efficiency

Tomakesureyourdifferentiationprotocoliseffective, compare the phenotype of your starting and treated cells withimmunocytochemistryorflowcytometry.Measurethe productionofsecretedproteinswithanimmunoassaythatdetects soluble proteins.

Immunoassay FAQs

Antibody Arrays

ELISA

ELISpot

FlowCytometry

Luminex®

Immune Cell Type Triggers, Maintenance Factors Transcription Factors Secretory Phenotype

ILC1 IL-12, -18 T-bet IFN-γ, TNF-α

ILC2 IL-4, -25, -33, TSLP GATA-3, RORα Amphiregulin, IL-4, -5, -9, -13

ILC3 IL-1α, -1β, IL-2, -7, -23 AHR, RORγT, T-bet IFN-γ, IL-17, -22

Regulatory ILC IL-2, TGF-β1 ID3, SOX4 IL-10, TGF-β1

Lti Cells IL-1β, -23 AHR, RORγT, RUNX1 IL-17, -22, Lymphotoxin

CytotoxicNKCells IL-12, -18 E4BP4, EOMES, T-bet Granzyme, Perforin

Th1Cells IFN-γ, IL-12, -18, -27 STAT4, T-bet IFN-γ, IL-2, TNF-α, TNF-β

Th2Cells IL-4 GATA-3, STAT6 IL-4, -5, -9, -13, -17E/25

Th9Cells IL-4, TGF-β1 BATF, GATA-3, IRF1, IRF4, PU.1, STAT6 IL-9, -10

Th17Cells IL-1β, -6, -21, -23, TGF-β1 RORγT IL-17A, -17F, -21, -22, -26

Th22Cells IL-6, TNF-α AHR, T-bet IL-10, -13, -21, -22, TNF-α

TfhCells IL-6, -21 Bcl-6, IRF4, STAT4 IFN-γ, IL-4, -10, -17, -21

TregCells IL-2, TGF-β1 FoxP3 Galectin-1, IL-10, -35, TGF-β1

Signal 1

Signal 2

TCR-CD3 ComplexMHCII

APC Naïve CD4+ T Cell

Th1

Th17

Th2

Th9

Th22

Tfh

Treg

Chapter 1 Chapter 2 Chapter 3 Chapter 4 Chapter 5 Chapter 6 Chapter 7 Chapter 8 Chapter 9 Chapter 10

6

Cell Type Triggers, Maintenance Factors Transcription Factors/Markers Secretory Phenotype

Ectoderm Noggin Otx2

Neural Progenitors EGF, FGFbasic, Noggin, SB 431542 Nestin, Pax6, SOX1

Cortical Neurons Noggin, Retinoic Acid, SB 431542 FoxG1, Pax6 pro-BDNF, Glutamate

Dopaminergic Neurons CHIR99021, FGF-8, Noggin, SB 431542, Shh HNF-3b/FoxA2, LMX1A, Msx1/2, TyrosineHydroxylase Dopamine

Spinal Motor Neurons Retinoic Acid, Shh CholineAcetyltransferase/ChAT, Islet-1, MNX1/HLXB9 Acetylcholine

Astrocytes BMP-2, CNTF, EGF, FGFbasic GFAP, NF-L, S100B, SOX9BDNF, G-CSF, GDNF, GM-CSF, IL-1β, IL-6, IL-8/CXCL8, NT-3, Thrombospon-din-1

Oligodendrocytes FGFbasic, IGF-I, LDN 193189, NT-3, PDGF, Retinoic Acid, SB 431542, Shh

CNPase, MBP, Oligodendrocyte Mark-er O4, Olig2

IL-6, Lipocalin-2/NGAL, MMPs, PDGF-AA

Endoderm Activin A, FGFbasic, Wnt-3a Claudin-7, HNF-3b/FoxA2, SOX7, SOX17

Hepatocytes Activin A, Dexamethasone, DMSO, EGF, FGF-4, FGF-10, HGF Albumin, HNF-4α/NR2A1

Albumin, SerpinA1/alpha1-Antitryp-sin, SerpinA3/alpha1-Antichymo-trypsin

Pancreatic Beta CellsActivin A, CHIR99021, FGF-10, HGF, IGF-I, KGF/FGF-7, LDN 193189, PDBu, Retinoic Acid, SANT-1

NKX6.1, PDX-1 Carboxypeptidase, IGF-II, Insulin

Lung Epithelium Activin A, BMP-4, CHIR99021, FGF-10, KGF/FGF-7, Retinoic Acid

NKX2-1, p63, SOX2 (proximal), SOX9 (distal)

Mesoderm BMP-4, FGFbasic Brachyury, Goosecoid, Hand1, Snail

Cardiomyocytes Activin A, BMP-4 TroponinT IL-10, TGF-β1

HematopoieticProgenitors Activin A, BMP-4, FGFbasic, Nodal, Notch-1, SCF, Shh, TGF-β1, VEGF, Wnt

CD34, CD43 (multipotential pro-genitors), Integrinalpha2b/CD41 (erythro-myeloid progenitors)

TCells Flt-3Ligand, IL-2, IL-7, SCF CD3, CD4, CD8 GM-CSF, IFN-γ, IL-2, TNF-α

NKCells Flt-3Ligand, IL-3, IL-15, IL-7, SCF FcgammaRIII/CD16, NCAM-1/CD56 Granzyme A, Granzyme B, IFN-γ, Perforin

Mesenchymal Stem Cells EGF, FGFbasic, PDGF-AB, TGF-β1 CD44, CD90, Endoglin/CD105 CCL5/RANTES, FGFbasic, HGF, IGF-I, IL-6, IL-10, MMPs, TGF-β, VEGF

Neural Cell Type

Triggers, Maintenance Factors

Astrocytes, Type1

CNTF, EGF, blocked by FGFbasic, PDGF

Astrocytes, Type2 Blocked by FGFbasic, PDGF

Cortical Neurons BDNF, IGF-I

Cortical Stem Cells EGF, FGFbasic

DRG Neurons β-NGF

GABAergic Neurons BDNF, GDNF, IGF-I, NT-4, Shh

Glutamatergic Neurons BMP-2, BMP-4, FGF-8b

HippocampalNeurons BDNF, IGF-I

Oligodendro-cytes CNTF, IGF-I, NT-3, PDGF

Spinal Motor Neurons

BDNF, CNTF, GDNF, IGF-I, Lamininα4

Cell Differentiation Posters

AstrocyteDevelopmentandFunction

B Cell Development

HematopoieticColonyIdentification

HematopoiesisandDevelopment

Innate Lymphoid Cell Lineage

Microglia Activation During Neuroinflammation

MouseTh17Differentiation

MouseandHumanDendriticCell Subsets

TCellSubsets

TheComplexBiologyofMacrophages

Unleash the Potential of Stem Cells

Interactive Pathways

Immunecelldifferentiationpathways

Stemcelldifferentiationpathways

Resources for Signal Transduction

Methods for detecting protein phosphorylation

ProfilingchangesinRTK phosphorylation video

Protocols

Immune cell differentiation protocols

Stem cell differentiation protocols

Interactive Cell Markers Tool

Thisisagreatresourceforcellsurface,secreted, and intracellular molecules. It covers immune, kidney, neural, and stem cells, plus subcellular organelles.

Fordifferentiationinformationonmorecelltypes,pleaseseethese additional resources.

Chapter 1 Chapter 2 Chapter 3 Chapter 4 Chapter 5 Chapter 6 Chapter 7 Chapter 8 Chapter 9 Chapter 10

7

Chapter 3 - Protein Interaction Studies

Chapter 4 - Protein Processing

Therearemanywaystostudyproteininteractionsincludingsurface plasmon resonance (SPR), ELISA type protein binding assay,co-immunoprecipitation,andfluorescenceresonanceenergytransfer(FRET).

Biotinylated proteinsarepowerfultoolsfordevelopingproteininteraction assays. Fluorokinereceptordetectionkits make use of biotinylated proteins to evaluate cell surface receptor expression.

Chemical derivatization of proteins is valuable for directed protein conjugation, including orientated protein coating on assayplatesandaffinitychromatographybeads.Itcanbeaccomplishedwithheterobifunctionalcrosslinkersthattargetspecificfunctionalgroupsonaminoacidsidechains.

Asyoucarryoutyourresearch,payattentiontohowyour protein of interest is produced, activated, and degraded. Amultitudeofproteasescleaveproteinswithdifferent recognition motifs, modes of enzymatic action, and biological results. PeptideCutter and MEROPS predict protein cleavage sitesintheaminoacidsequenceyouprovide.Smallmoleculeprotease inhibitors can help you control various steps in the processing of your protein.

Signal Peptide Cleavage

Secreted proteins and many transmembrane proteins are translatedwithanN-terminalsignalpeptidewhichdirectsthenascentproteinthroughthesecretorypathway.Thesepeptidesare cleaved by signal peptidases. SignalP-5.0 can predict sig-nal peptide cleavage sites in your protein.

Propeptide Removal

Manyproteinsareexpressedwithapropeptidethatkeepsthem inactive (e.g. enzyme zymogens). Proprotein activation can be carried out by many enzymes including proprotein con-vertasessuchasFurin.Propeptidecleavageisrequiredfortheactivationofmetalloproteases(MMPs),BMPs,Renin,andHGF.

Our Custom Servicescanhelpyouwithspecialtyprotein conjugations and constructs for your protein interaction research.

Watch our video to learn more about protein biotinylation.

Chapter 1 Chapter 2 Chapter 3 Chapter 4 Chapter 5 Chapter 6 Chapter 7 Chapter 8 Chapter 9 Chapter 10

8

Protein Activation

Thebloodcoagulationpathwayinvolvesacascadeofserineproteaseswhichbecomeactivatedfollowingcleavagebyanupstreamprotease.Thefinalstepofclotformationistheproteolyticconversionoffibrinogentofibrin.Thecomplement pathway is also a cascade that involves multiple proteases.

Many peptide hormones, such as Insulin and Endothelin, requireproteolyticactivation.Insomecases,cleavageofa single precursor protein can release several peptide hormones, asseenwithPOMCcleavageleadingtothegenerationofACTH,MSH,beta-Endorphin,Met-enkephalin,and Melanocortin. Cleavage of Angiotensinogen by multiple proteases gives rise to over a dozen Angiotensin peptides withdistinctactivities.

Some proteins (e.g. the chemokine CXCL5) can be processed tovariouslengthswiththeshortenedformsexhibitingmodifiedbioactivity.

Theintracellulardomain(ICD)ofcertaintransmembrane proteinscanbecleavedinaprocessknownasregulated intramembrane proteolysis (RIP). In RIP, cleavage liberates the ICD to translocate to the nucleus and regulate gene transcription. Examples of ICD cleavage include Notch family proteins and Amyloid Precursor Protein (APP).

Antigen presentation is important for immune system recognition and relies on proteolysis for the formation of antigenic peptides. Intracellular proteins or proteins internalized by antigen presenting cells (APC) are cleaved intopeptideswhichassociatewithMHCmoleculesonthe cell surface.

Protein Degradation

Studythedegradationofyourproteinbyproteasesthatworkby different mechanisms: aspartic proteases, serine proteases, cysteine proteases, metalloproteases, or lysosomal enzymes.

Theintracellulardegradationofyourproteinmaybetriggeredby the addition of ubiquitin or SUMO molecules in various linkages. E3ubiquitinligases and deubiquitinatingenzymes(DUBs)carryoutubiquitin-proteinmodificationsandmay regulate the intracellular turnover of your protein.

PROTACs® are bifunctional compounds that couple an activatedE3ubiquitinligasewithatargetproteintoinducethetargetprotein’subiquitinationandintracellulardegradation.

Protease Assay Protocols

ACE-2, human

ACE-2, mouse

BMP-1/PCP

Meprin 1a

FluorogenicPeptideSubstrates

MMP Assay Principle

UbiquitinProtocols

Protease activity assays and reagents, substrates and inhibitors.

Interactive Pathways

Autophagy

Ubiquitination

Bloodcoagulationinteractivepathway

Renin-Angiotensininteractivepathway

Chapter 1 Chapter 2 Chapter 3 Chapter 4 Chapter 5 Chapter 6 Chapter 7 Chapter 8 Chapter 9 Chapter 10

9

Chapter 5 - Antibodies and Immunoassays

Immunogen Preparation

Correctly folded and bioactive proteins willserveyoubestforraising,purifying,andcharacterizingantibodies.They’llgive you the best chance of obtaining antibodies to your protein in its native conformation. Conjugate your protein to polymers like polyethylene glycol (PEG) to increase its circulating half-life. Boost the immuneresponsebyaddinginFreund’sadjuvants or individual molecules that targetspecificimmunesystemmoleculesor cell types.

Vaccine Development

Formulateyourproteinoracocktailofproteins into a vaccine. It’s a similar principle to designing an immunogen withtheaddedgoaloftriggeringaresponse that leads to in vivo immune protection against pathogens.

Antibody Purification

Ifyouwanttoisolateantibodiesfromserum or cell culture media, prepare a purificationcolumnbyconjugating bacterial Protein A or Protein G to the chromatographyresin.Theseproteinsbind the heavy chains of most antibodies, so you can recover the entire immunoglobulin pool.

Ifyouwanttoaffinitypurifyanantibodyfrom a polyclonal mixture, conjugate your immunizing protein to the beads and recover just those antibodies that bind theantigen.Thismethodisalsoeffectivefor clearing antibodies that are a major component of serum (either from the immunized host or that you added to your cell culture).

Immunoassay Development

Tobeabletoobtainquantitative numbers from an immunoassay, generateastandardcurvewithyourprotein.Thiswillalsoletyoudeterminethe sensitivity and linearity of your assay. With a standard curve, you can convert the assay’s signal (optical density or band intensity) to an approximate protein concentration in the sample.

Ahighlypurified,biologicallyactiveprotein is a good positive control in your immunoassay.

Biotinylatedproteinsarepowerfultoolsfor developing protein immunoassays because of their extremely tight binding to streptavidin. Biotin can be coupled to variousfunctionalgroupsinproteinswithheterobifunctional crosslinkers. Site-specificbiotinylationcanbe accomplishedwithAvi-tag biotinylated proteins that make use of enzymatic biotinylationinaconsensussequence.

Antibody Characterization

Proteinsarevitalforconfirmingthe antigenspecificityofanantibody. Immunoassays such as Western blot and ELISA provide information about antibody cross-reactivitywithrelatedproteins.Theycanhelpyoudeterminehow selectively an antibody recognizes your protein compared to structurally related proteins or orthologs from other species.

Forsomebasicinformation on antibody production and purification,visitPrimary Antibody Selection and Optimization.

Immunoassay Resources

What is an ELISA?

What is the Importance of ELISA Controls?

10 Best ELISA Practices and Techniques

ELISAFAQs

TroubleshootingGuide:ELISA Development

WesternBlotTroubleshootingGuide

TMBSubstrate

TMBSubstrate

Stop

HRP

Chapter 1 Chapter 2 Chapter 3 Chapter 4 Chapter 5 Chapter 6 Chapter 7 Chapter 8 Chapter 9 Chapter 10

10

Chapter 6 - Protein Characterization

Protein Biological Activity

Tomakesuretheproteinsyouusearefunctional,youshouldhaveareliableassay.Youmightneedtoconfirmyourproteinshave enzymatic activity, trigger receptor signaling, regulate cell-cell adhesion, or serve as a carrier for small molecules.

Learn more about measuring protein biological activity, includingthebestwaytocomparethebioactivityofproteinsfromdifferentvendors.Thisunit conversion tableshowshowwederiveouractivityvalues(units/mg) and correlate them to WorldHealthOrganization(WHO)standards.

Ifyou’reinterestedinenzymesubstratespecificityandkinetics,the data sheet for each of our recombinant enzymes includes a straightforwardassayprotocol.

Crystallize your protein for X-ray diffraction studies to generate a 3-dimensional model of its structure. Use the crystal structureasthebasisofstructure-functionstudiesthatwillhelpyoudefinewhichregionsoftheproteinareinvolvedindifferentfunctions.Thesefreeprogramsarevaluablefor displayingmoleculecoordinatefiles(pdbfiles)availablefromthe Protein Data Bank.

RasMol

Swiss-pdbviewer

PyMOL

Activity Assay FAQs

AnnexinV,Caspase,Phosphatase,andTUNEL Assays.

Chapter 1 Chapter 2 Chapter 3 Chapter 4 Chapter 5 Chapter 6 Chapter 7 Chapter 8 Chapter 9 Chapter 10

11

Chapter 7 - Tissue Regeneration

Artificial Tissue Formation Thisisarapidlydevelopingfieldwithapplicationsinregenerativemedicine,transplantation,andwoundhealing.Artificialtissuesand extracellular matrix have applications in grafting (skin, bone, vasculature, etc) or to enhance the engraftment of natural tissues and organs.

ArtificialtissuesareoftenproducedbyseedingcellsintoamatrixstructureofdefinedECM molecules, proteoglycans, or decellularizednaturaltissues.Tissuedecellularizationinvolvesenzymaticorchemicaldigestionand/orphysicaldisruptionofthecellularcomponentofatissue.Theremainingextracellularmatrixshouldretainthenativeshapeforthetissuewhichwillfacilitateproperfunctioningoftheartificialtissue.

Artificialmatrixcanbechargedwithawidevarietyofreleasablebioactiveproteinstoimprovethesuccessofengraftment.Inparticular, angiogenic factors, bone morphogenetic proteins (BMPs), or other cytokinesandgrowthfactors can directly promote tissuegrowthorregulatetheinfiltrationofimmunecells.

Organoidsand3-dimensionalcellculturecanbeusedasmodelsystemsforstudyingtoxicology,drugefficacy,organ development,anddiseaseprocessesliketumorprogression.They’retypicallysupportedbybiologicalorsyntheticmatrix scaffoldsthatpromotethespatialorganizationofdifferentcelltypes.Theseculturesmimictissuearchitectureinwaysthat simple cell culture can’t. If you build an in vitro system for these uses, you can reduce the expense and variability of using live animals.

Organoid Recipes Organoid Videos Organoid and 3-D Cell Culture Protocols

Chapter 1 Chapter 2 Chapter 3 Chapter 4 Chapter 5 Chapter 6 Chapter 7 Chapter 8 Chapter 9 Chapter 10

Organoid Resources

12

Chapter 8 - Bioprocessing EnzymesManyenzymesarevaluableinthebioprocessingworkflow,fromnucleasesthatcleanupyourcellcultureharvestmaterialtoenzymesthattreatyourproductforanalysisorpurification.

EvengentleharvestingofcellculturesresultsinthelysisofsomecellsandthereleaseofgenomicDNA.ThisDNAwillinterferewithsubsequentproductpurificationstepsandshortenthelifespanofchromatographyresins.TreatingyourcellcultureharvestwithnucleasescanlimitthedetrimentaleffectsofDNAondownstreambioprocessingstages.

Enzymes that cleave glycansfromglycoproteinsfacilitatetheanalysisofthoseglycanswhichisimportantforproduct characterization.Inparticular,PNGaseF,Heparinases,andChondroitinasescomplementeachotherwiththeirdistinct specificitiesofglycanremoval.

Glycan synthesis enzymes, including sialyltransferases, fucosyltransferases, GlcNAc transferases, and GalNAc transferases areusefulforthelabeling,modification,andimagingofspecificlinkagesofglycans.Theseassayscanbevaluableforbiologic characterization and evaluating product consistency.

ProteasessuchasPGPEP-1arekeyforthecharacterizationofproteinbiologics.Theycanbeusedtogeneratepeptidesfor analysisandforEdmandegradationduringN-terminalsequencing.

Ifyourproteinhasapeptidetagordomainforexpression,purification,ordetectionpurposes,youmayneedtoremoveitmid-process.Thesetagsareconnectedtotheproteinwithaproteasesensitivelinkerthat’stypicallycleavedby Enteropeptidase/Enterokinase,CoagulationFactorXa,TEVProtease,Thrombin,orUsp1.His-tags,Fcfusionproteins,SUMO,GST,Flagfusionproteins,andpropeptidefusionsarecommonlyusedconstructs.Toconfirmthatyou’veremovedthetag,useanepitope tag antibody.

Highlypurifiedproteinsarevaluableasstandardsatmanypointsalongthebioprocessingworkflow.You’llneedthemtodevelop and monitor the performance of each step, and also to test the integrity and functionality of the product itself.

DownloadacopyofourBioprocessing brochure to learnaboutallthewaysthatproteintoolsaddto thebioprocessingworkflow.

Chapter 1 Chapter 2 Chapter 3 Chapter 4 Chapter 5 Chapter 6 Chapter 7 Chapter 8 Chapter 9 Chapter 10

13

Chapter 9 - In vivo Animal and Human StudiesIf you’re carrying out in vivo research, consider using reagents producedwithbetterdefinedcompositionandformulationthan common research use only (RUO) reagents. Animal-Freerecombinant proteinsaremanufacturedwithoutcellcultureserum or other animal products, and they’re manufactured withanincreasedlevelofprocesscontrolsand documentation.

If you’re intending to advance from animal models to human studies, it makes sense to adopt GMP-grade proteins early in theprocesssoyoudon’thavetovalidatenewreagentswhenthings get more complicated. A high level of control and documentationisrequiredforproteinsusedasancillary componentsduringclinicalmanufacturing.Thisrequirementis met by GMP-grade proteinswhichareaccompaniedbyGMP proteincertificatesofanalysis. GMP-grade proteins are producedinaccordancewithFDA-approvedqualitypolicyandregulatory support. With the added rigor, you satisfy the regulatoryrequirementsandyougetincreasedconfidenceinthe purity and integrity of the protein.

Developingimmunoassaysforcommercialdiagnosticsrequiresproteins that are produced in a tightly regulated and thoroughly documented environment. Both R&D Systems and BiosPacific areexperiencedinprovidingthesequalifiedreagents.

AnimalComponent-Free Process Statement

GMPproteinFAQs

GMP proteins brochure

Chapter 1 Chapter 2 Chapter 3 Chapter 4 Chapter 5 Chapter 6 Chapter 7 Chapter 8 Chapter 9 Chapter 10

14

Chapter 10 - General ResourcesProtein Specific Activity

Potencyandenzymaticactivityaredefinedonthebasisofactivityunitsper microgramofprotein.Comparisonoftwoproteinpreparationsonthebasisof massalonedoesnottakeintoaccountthefunctionalstateofaprotein.Sequence, formulation,PTM,purity,andoligomerizationcanalsohaveconsiderableeffectson specificactivity.OurUnitConversionTableshowsthecorrelationofselect R&DSystems®cytokinesandgrowthfactorswithWorldHealthOrganization(WHO) standards.Italsoshowsthecelllineusedforevaluationbybioassay.Reliable comparisonofreportedactivityvaluesrequiresthatthesameassayisperformed for both samples side by side.

Protein Detection Methods

Whataretheprosandconsofdifferentproteinquantitationmethods?Absorbanceat280nmletsyouquantitatethetotalamountofproteininasample.It’smeasuredwithaspectrophotometeranddependsontheaminoacidcontentofyourprotein(primarilytryptophan).Extinctioncoefficientsareexperimentallydeterminedfor individual proteins and let you correct A280 readings for your protein of interest. Thesemeasurementscanbeelevatedforproteinscontainingcofactorsorbuffercomponents that absorb near 280 nm.

Coomassie/Bradfordassaysalsoquantitatethetotalamountofproteininasample,withaspectrophotometersettoreadabsorbanceat595nm.TheCoomassiedyereactsprimarilywitharginine,lysine,andhistidineresidues.Coomassiebrilliantblueis commonly used for detecting proteins separated by SDS-PAGE.

TheBCAassay(bicinchoninicacid)alsomeasurestotalproteininasample.Itcanbea very useful alternative method, particularly in the presence of buffer components thatinterferewiththeBradfordassay.

Immunoassayscanquantitatetheamountofaparticularproteininasample.TheyaremorespecificthanA280andCoomassieassays,becausetheyonlydetect protein that’s recognized by the antibody used. With a standard curve, you can quantitatethespecificproteininthepresenceofcontaminating proteins.Immunoassayaccuracydependsonthespecificityofantibodiesyouuse.

Freeze/Thaw and Aliquotting

Yourproteinmaydenatureorprecipitateifyoufreezeandthawitrepeatedly. Aliquottingitintosmallervolumeswillreducethenumberoftimesyouhavetofreezeandthawyourproteinsolution.Detergents,glycerol,orcarrierproteinsuchasBSAcanimprovestabilityandlimittheamountofproteinyoulosebynonspecificbindingtothewallofyourvialsandtubes.

Questions about Proteins and Enzymes?

ProteinsandEnzymesFAQs

RecombinantProteinsFAQs

If you have a different question,pleaseaskour TechnicalServiceexpertswhocanfindtheanswertomostanyproteinrelatedquestion!

A280=(extinctioncoefficient)x (molar protein concentration) for a 1 cm path length.

See our Peptide Nomenclature Guide for amino acid structures, uncommon amino acids, and substituents.

Molarity and Reconstitution Calculators

Wehavequickandeasytools to simplify your calculations.

If you’d like to learn more about reconstitution of lyophilized proteins,watchourvideo.

Chapter 1 Chapter 2 Chapter 3 Chapter 4 Chapter 5 Chapter 6 Chapter 7 Chapter 8 Chapter 9 Chapter 10

15

Cross-Functionality

Quiteoften,proteinsofonespeciesareactiveoncellsorbindtomoleculesfromadifferentspecies.Sometimes,though, even100%aminoacidsequenceidentityisn’tenoughtoconfercross-functionality.Thisislikelyduetodifferencesin post-translationalmodifications.Cross-functionalityisapropertythatappliestoproteinactivity.Thisisnotthesameas cross-reactivitywhichreferstotheabilityofanantibodytorecognizedistinctmoleculesorepitopes.

Post-Translational Modifications

PTMsareadditionsoralterationstotheprimaryaminoacidsequenceofaprotein.Themodificationsarecarriedoutbyenzymes that target recognition motifs in the protein, and they canbefurthermodifiedbyadditionalenzymes(asinthe multi-step process of protein glycosylation).

Prediction and Computation Tools

TheExPASy Bioinformatics Resource Portal provides an extensivecollectionoffreeproteinsequenceanalysis algorithms.Here’saselectlistforcommonlyqueriedfeatures.

Protein Modifications PTM Enzymes

Acetylation acetyltransferases and deacetylases

Acylation prenyltransferases

Citrullination peptidylarginine diminases (PAD)

Glycosylation (N- and O-linked)

glycosyltransferases and glycosidases

Hydroxylation pro-oxidant and anti-oxidant enzymes

Methylation methyltransferases and demethylases

Phosphorylation (Ser/ThrandTyr)

intracellular kinases and phosphatases

SUMOylation E1 activating enzymes, E2 conjugating enzymes, E3 Ligases

TyrosineSulfation sulfatases and sulfotransferases

Ubiquitination E1 activating enzymes, E2 conjugating enzymes, E3 Ligases

Carbamylation chemicalmodification

Glycation chemicalmodification

S-nitrosylation Nitrosylating Molecules

PTM Prediction Algorithm

Acetylation site (N-terminal) NetAcet

Glycosylation site (N-linked) NetNGlyc 1.0

Glycosylation status GlycoDomainViewer

GPI-anchor signal GPI-SOM

GPImodificationsite Big-PI

Myristoylation site (N-terminal) Myristoylator

Mucin type GalNAc O-glycosylation site NetOGlyc

Phosphorylationsite(Ser,Thr,Tyr) NetPhos 3.1

Prenylation site PrePS

Tyrosinesulfationsite Sulfinator

Tyrosinesulfationsite SulfoSite

Sequence Alignment, Cleavage, and Structure

pI,molecularweight,E280 ProtParam

Signal peptide cleavage site SignalP-5.0

Transmembranetopologyandsignalpeptide Phobius

Secondary structure CFSSP

Coiled coil region COILS

Protease cleavage site PeptideCutter

Protease cleavage site MEROPS (from EMBL-EBI)

Peptides from protein cleavage PeptideMass

Sequencesimilaritysearch BLAST

Sequencealignment ClustalW

Multiplesequencealignment Clustal Omega

Protein Structure Visualization

RasMol | Swiss-pdbviewer | PyMOL

Ubiquitin Protocols

Conjugation,poly-vs.mono-ubiquitination,andchainlinkages.

Glycobiology White Paper

Withprotocolsforspecificglycanlabeling, visualization,andmodification.

Chapter 1 Chapter 2 Chapter 3 Chapter 4 Chapter 5 Chapter 6 Chapter 7 Chapter 8 Chapter 9 Chapter 10

16

1st base

2nd base 3rd baseU C A G

U

UUU(Phe/F)Phenylalanine

UCU

(Ser/S)Serine

UAU(Tyr/Y)Tyrosine

UGU(Cys/C)Cysteine

U

UUC UCC UAC UGC C

UUA

(Leu/L)Leucine

UCA UAA Stop (Ochre) UGA Stop (Opal) A

UUG UCG UAG Stop (Amber) UGG (Trp/W)Tryptophan G

C

CUU CCU

(Pro/P)Proline

CAU(His/H)Histidine

CGU

(Arg/R)Arginine

U

CUC CCC CAC CGC C

CUA CCA CAA(Gln/Q)Glutamine

CGA A

CUG CCG CAG CGG G

A

AUU

(Ile/l)Isolucine

ACU

(Thr/T)Threonine

AAU(Asn/N)Asparagine

AGU(Ser/S)Serine

U

AUC ACC AAC AGC C

AUA ACA AAA(Lys/K)Lysine

AGA(Arg/R)Arginine

A

AUG[A] (Met/M)Methionine ACG AAG AGG G

G

GUU

(Val/V)Valine

GCU

(Ala/A)Alanine

GAU(Asp/D)Asparticacid

GGU

(Gly/G)Glycine

U

GUC GCC GAC GGC C

GUA GCA GAA(Glu/E)Glutamicacid

GGA A

GUG GCG GAG GGG G

How Can We Help?

Custom protein services for protein engineering including

• Point mutations

•Fusionproteins

• Direct-labeled proteins

• Dual function proteins

Search for your protein of interest Contact us for customer service and technical service.

Bulkquoterequest

DW_ProteinseBook_RSDFY20-12138

Globalinfo@bio-techne.combio-techne.com/find-us/distributorsTEL+16123792956North AmericaTEL8003437475Europe | Middle East | AfricaTEL+44(0)1235529449Chinainfo.cn@bio-techne.comTEL+86(21)52380373

bio-techne.com

RnDSy-2945 Novus-cmyk Tocri-2945

Prote_2945

Forresearchuseormanufacturingpurposesonly.Trademarksandregisteredtrademarksarethepropertyoftheirrespectiveowners.©2018Bio-TechneCorporation

Chapter 1 Chapter 2 Chapter 3 Chapter 4 Chapter 5 Chapter 6 Chapter 7 Chapter 8 Chapter 9 Chapter 10

Genetic Code