Transcription of Bioconjugation technical handbook
1 Bioconjugation technical handbook Reagents for crosslinking, immobilization, modification, biotinylation, and fluorescent labeling of proteins and peptidesBioconjugation is the process of chemically joining two or more molecules or biomolecules by a covalent bond. This technique utilizes a variety of reagents for the crosslinking, immobilization, modification, and labeling of proteins and peptides. Bioconjugation reagents contain reactive ends to specific functional groups ( , primary amines, sulfhydryls) on proteins or other molecules. The availability of several chemical groups in proteins and peptides make them targets for a wide range of applications, including biotinylation, immobilization to solid supports, protein structural studies, and metabolic labeling.
2 Chemical agents may be used to modify amino acid side chains on proteins and peptides in order to alter charges, block or expose reactive binding sites, inactivate functions, or change functional groups to create targets for crosslinking and labeling. Crosslinking, labeling, and modification reagents can be described by their chemical reactivity, molecular properties, or applications (Table 1).IntroductionPackaging optionsSelect a package size or grade based on the scale of your reaction or your requirements. These reagents are available from milligram to kilogram to gramMilligram to gramGram to kilogramBulk-sizepackagesavailableSingle - tubes Catalog productPremium gradeLarge-volume or custom packagesMolecular propertiesSecond, choose which features or characteristics are important for your modificationsNOOOOONOOODSSD isuccinimidyl suberateMW arm same reactivity on both endsDSP with disulfide linker for cleavageTCEP reduces disulfide bondsSpacer arm compositionSpacer arm lengthSpacer arm Arm Arm OOONNOOOn = 8CA(PEG)
3 8MW Arm n = 12CA(PEG)12MW Arm n = 24CA(PEG)24MW Arm OOHONH2nCA(PEG)4MW Arm OOOOOHONH2CA(PEG)nCarboxy-PEGn-amineCarb oxyl-(ethyleneglycol)n BMH with hydrocarbon spacerAMAS short spacer between reactive groupsCA(PEG)n adds solubility in aqueous solutionsTable 1. Key considerations for selecting the right Bioconjugation reactivityFirst, select a reagent with the functional group(s) to bind your biomolecules of ester reactionMaleimide reactionAmine-containingmoleculeNHS estercompoundAmine bondNHSS ulfhydryl-containingmoleculeMaleimidecom poundThioether bondpH 7.
4 5 ApplicationsSelect the specific reagent depending on the application ( , protein detection, immobilization, or interaction studies).LabelingImmobilizationProtein interaction studiesHOOOSHNNHNOOOOONOOODSSD isuccinimidyl suberateMW arm NSOOOOONOOOODSSOD isuccinimidyl sulfoxideMW Arm Biotin for labeling and detectionDSS couples proteins to surfacesDSSO MS-cleavable crosslinkerChemical reactivity of Bioconjugation reagents Introduction 5 Amine-reactive chemical groups 6 Carboxylic acid reactive chemical groups 7 Sulfhydryl-reactive chemical groups 8 Carbonyl-reactive chemical groups 10 Nonspecificly-reactive chemical groups 12
5 Chemoselective ligation 13 Molecular properties of Bioconjugation reagents Introduction 14 Homobifunctional and heterobifunctional crosslinkers 15 General reaction conditions 16 Modifications 16 Spacer arm length 18 Spacer arm composition 18 Spacer arm cleavability 18 Spacer arm structure and solubility 19 Applications using Bioconjugation reagents Introduction 20 Protein and peptide biotinylation 21 Fluorescent labeling of proteins and peptides 23 Protein immobilization onto solid supports 25 Surface modification using PEG-based reagents 26 Hapten carrier conjugation for antibody production 27 Protein protein conjugation 28 Creation of immunotoxins 29 Label transfer 29 Subunit crosslinking and protein structural studies 31 Protein interaction and crosslinking using mass spectrometry 32MS-cleavable crosslinkers 33In vivo crosslinking 34 Metabolic labeling 35 Cell surface crosslinking 36 Cell membrane structural studies 36 Special packaging to meet specific
6 Bioconjugation needs Introduction 37No-Weigh packaging format for Bioconjugation reagents 38 Premium-grade Bioconjugation reagents 39 Bioconjugation resources 40 Glossary 42 References 44 Ordering information 50 Related handbooks and resources 71 Contents5 Chemical reactivity of biconjugation reagentsIntroductionThe most important property of a Bioconjugation reagent is its reactive chemical group.
7 The reactive group establishes the method and mechanism for chemical modification. Crosslinkers contain at least two reactive groups, which target common functional groups found in biomolecules such as proteins and nucleic acids. Protein modification reagents like PEGylation or biotinylation reagents have a reactive group at one terminus (PEG chain or biotin group, respectively) and a chemical moiety at the other end. The functional groups that are commonly targeted for Bioconjugation include primary amines, sulfhydryls, carbonyls, carbohydrates, and carboxylic acids (Figure 1, Table 1).
8 Coupling can also be nonselective using photoreactive acidRFigure 1. Common amino acid functional groups targeted for 2. Popular crosslinker reactive groups for protein classTarget functional groupReactive chemical groupAmine-reactive NH2 NHS ester Imidoester Pentafluorophenyl ester Hydroxymethyl phosphineCarboxyl-to-amine reactive COOHC arbodiimide ( , EDC)Sulfhydryl-reactive SHMaleimide Haloacetyl (bromo-, chloro-, or iodo-) Pyridyl disulfide Thiosulfonate Vinyl sulfoneAldehyde-reactive ( , oxidized sugars, carbonyls) CHOH ydrazide Alkoxyamine NHS esterPhotoreactive ( , nonselective, random insertion)RandomDiazirine Aryl azideHydroxyl (nonaqueous)
9 -reactive OHIsocyanateAzide-reactive N3 Alkyne PhosphineAmine-reactive chemical groupsPrimary amines ( NH2) exist at the N-terminus of each polypeptide chain (called the -amine) and in the side chain of lysine (Lys, K) residues (called the -amine). Because of their positive charge at physiological conditions, primary amines are usually outward facing ( , on the outer surface of proteins), making them more accessible for conjugation without denaturing protein structure. A number of reactive chemical groups target primary amines (Figure 2), but the most commonly used groups are N-hydroxysuccinimide esters (NHS esters) and imidoesters.
10 NRCSRNONOONHOI sothiocyanateIsocyanateAcyl azideNHS esterRSClOOSulfonyl chlorideOHRA ldehydeROEpoxideR OOROC arbonateFRFluorobenzeneImidoesterCH3 ONH2 RNNCNHClCarbodiimideFluorophenyl esterFFFOFFROOOORA nhydrideNRCOF igure 2. Reactive chemical groups that target primary esters (NHS esters)NHS esterOONOORNHS esters are reactive groups formed by EDC activation of carboxylate molecules. NHS ester activated crosslinkers and labeling compounds react with primary amines in slightly alkaline conditions to yield stable amide bonds (Figure 3). The reaction releases N-hydroxysuccinimide, which can be removed easily by dialysis or chemistryNHS ester crosslinking reactions are most commonly performed in phosphate, carbonate bicarbonate, HEPES, or borate buffers at pH for 30 minutes to 4 hours at room temperature or 4 C.
