Conjugates Labels Tags
Naked proteins have proven their value as successful biopharmaceuticals, still they suffer from some limitations. In order to improve expression levels, solubility, folding, purification and detection of recombinant proteins, a very strategy is the conjugation with peptides or proteins. Most protein and cell analysis techniques involving antibodies rely on conjugates to enhance or simplify the procedure. The formation of a stable, covalent linkage will often be done chemically, but it can also be performed biochemically.
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The conjugate choice depends upon the experimental application and therefore the and allows colorimetric, fluorescent or chemiluminescent detection of primary antibodies in cell imaging, immunohistochemistry, flow cytometry and western blotting. antibodies-online offers conjugated antibodies linked to a variety of colorimetric and fluorescent labels for use in detection, purification, sorting, and microscopy applications.
For the seperation of proteins a variety of conjugates can be used with a high specificity for a certain matrix. Those conjugates are often short 10-15 amino acid sequences and referred to as tags. Because these tags are entities with known sequences and well characterized physicochemical properties, they are an essential tool in molecular biology for purification of recombinant proteins. Epitope tags are usually engineered onto either the N- or C- terminus in order to minimize tertiary structure disruptions that may alter protein function. Well-known examples are GST tag, HIS tag and the interaction between biotin and streptavidin.
The detection and localization of an antigen is next to separation another important task done by tags. Fluorescent tagging is a predominant technique ensuring a visual readout. The non-fluorescent conjugates can be divided into chemical and biological compounds. Biological active compounds will often be used for colorimetric detection of the antigen, for example for western blotting. If the immunolabeling process is meant to reveal information about isolated cells or its substructures, the process is called immunocytochemistry, immunohistochemistry generally refers to experiments where targets within thin sections of tissue are stained.
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A fluorescent tag, also known as label, is attached chemically to the protein, antibody, or amino acid aid in order to label and detect the biomolecule to a particular site within a cell, tissue, or organ. Generally, fluorescent tagging, often referred to as immunolabelling, uses a reactive derivative of a fluorescent molecule known as a fluorophore, a class of light absorbing and light emitting molecules. GFP and its variants are the most commonly used fluorescence tags.
A single dye is excited at a particular wavelength and emits a photon at a higher wavelength. Fluorescence detection is based on the difference of maximal absorption and emission for every fluorophore, defined as Stokes shift. A large Stokes shift is indicative of less overlap between the two wavelengths, and is highly desirable since it means that the emitted light can be clearly distinguished from the light source which was used for excitation. The range of fluorescent tags is constantly growing - new alternatives offer higher extinction coefficient, quantum yield or photostability which enables brigther signals and multiplexing capabilities.
Tandem dyes provide added benefit, as they allow for more markers to be evaluated at once especially in multicolor panel experiments. The tandem dye consists of two different covalently attached fluorophores, a donor and an acceptor molecule. When placed in close proximity energy is passed on from an excited donor to the acceptor molecule. This happens via Förster resonance energy transfer (FRET), a mechanism describing energy transfer between two light-sensitive molecules.
The tandem dye is excited at the excitation wavelength of the donor molecule and emits a photon at the emission wavelength of the acceptor molecule.
- Avoid photobleaching via light exposure
- Do not freeze to minimize denaturation
- Be aware of cross-beam excitation
- Do not fix longer than 30 minutes
- High batch-to-batch variation
- Narrow emission spectra minimize bleed-through
Using tandem dyes, a single laser can excite several fluorophores, which are measured by different detectors. This improves multicolor flow cytometry by a big margin, 12 and more color flow cytometry panels are viable.
Additionally the Stokes shift of a tandem dye is greater than that of the original donor molecule leading to better distinction between excitation source and emission. Therefore avoid overlapping emission spectrums for co-localization experiments of two different proteins. When performing multi-color immunofluorescence experiments, use fluorophores with narrow emission spectra, this way bleed-through is minimized as well.
Fluorescent Conjugated Antibodies, Kits and Proteins
|Fluophore||Excitation Peak (nm)||Emission Peak (nm)||Emission Color|
|Alexa Fluor 405||401||421|
|Alexa Fluor 350||346||442||ATTO 390||390||470|
|Alexa Fluor 488||493||519|
|Alexa Fluor 594||550||570|
|Alexa Fluor 647||651||667|
|Alexa Fluor 700||702||723|
|Alexa Fluor 790||784||814|
Epitope Tags & Fusion Proteins
|Name||Sequence||Typical use(s) *||Comments|
|AP Tag||444 AA||D, Purif||Colorimetric detection; useful for Western bloting, far-Western blotting, southern blotting, sandwich ELISA, and subcellular localization in bacterial and mammalian expression systems; convenient purification of crude periplasmic extract from bacteria; antibody purification does not give high yields; low pH elution may irreversibly affect protein properties; matrix is of limited reusability; AP dimerization and large size may affect properties of fusion.|
|AU1 Tag||DTYRYITDFYLK||D, Purif||Used for visualization by immunochemical methods. The recognized AU1 sequence represents the amino acid sequence DTYRYI, derived from the major capsid protein of bovine papillomavirus-1 (BPV-1). The recognized AU5 epitope represents the amino acid sequence TDFYLK.|
|AU5 Tag||DTYRYITDFYLK||D, Purif|
|AviTag||GLNDIFEAQKIEWHE||Func, SDS, ELISA, WB||The patented AviTag™ technology employs a highly targeted enzymatic conjugation of a single biotin on a unique 15 amino acid peptide tag in vitro or in vivo, using the biotin ligase (BirA) from E. coli. Purify AviTag-fusion proteins using monomeric avidin. Visualize AviTag-fusion proteins using avidin or streptavidin conjugates with western blots and MHC-tetramers for staining and sorting T cells.|
|Biotin Ligase Tag||GLNDIFEAQKIEWH||Detection and purification||The Biotin Ligase tag is described as the minimal peptide sequence that serves as a the substrate for the E. coli biotin holoenzyme synthetase, BirA. In the presence of active BirA, the biotin ligase tag (also known as the biotin acceptor peptide) becomes biotinylated, allowing for detection or purification with streptavidin.|
|CBP Tag||MKRRWKKNFIAVSAANRFKKISSSGAL||WB, FACS, IHC, IP, IF||Calmodulin Binding Protein (CBP) affinity tag is a useful tag in protein study which can be added to the N or C terminus of proteins of interest through DNA recombinant technology. The tag is derived from muscle myosin light-chain kinase. The tag comprises 26 amino acid residues with the molecular weight of 4 kDa. CBP tag has the relatively high affinity for calmodulin (CaM). CBP-tagged proteins can be purified from crude cell extracts through CaM affinity resin.|
|CaM Tag||KRRWKKNFIAVSAANRFKKISSSGAL||WB, FACS, IHC, IP, IF|
|FLAG® Tag||DYKDDDDK||Func, PI, WB, IP, AbP||The FLAG® Expression System is a proven method to express, purify and detect recombinant fusion proteins. It utilizes a short, hydrophilic 8-amino acid peptide likely to be presented at the protein surface. The easy access simplifies antibody binding as well as cleavage by enterokinase (Ek). Small size of the FLAG® peptide tag does not obscure other epitopes, domains, or alter function, secretion, or transport of the fusion protein. Sensitivity can be enhanced using the 3x FLAG® epitope.|
|glu-glu Tag||EYMPME||WB, ELISA, ICC/IF, IP, Purif||The glu-glu epitope tag is commonly engineered onto the N- or C- terminus of a protein of interest so that the tag protein can be analyzed and visualized using immunochemical methods. The recognized glu-glu epitope represents amino acid residues 314-319, EYMPME, of middle T antigen of mouse polyomavirus.|
|GST Tag||Fusionsprotein 220 AA||AP||A protein which binds to immobilized glutathione GST (glutathione S-transferase). It is commonly engineered onto the N- or C- terminus of a protein of interest to create fusion proteins that can be purified in a pull-down assay, visualized and analyzed via the properties of the GST tag.|
|HA Tag||YPYDVPDYA||SDS, IHC, WB, ELISA||The HA (human influenza hemagglutinin) epitope tag is a surface glycoprotein required for the infectivity of the human virus. The HA tag is derived from the HA-molecule corresponding to amino acids 98-106. It does not appear to interfere with the bioactivity or the biodistribution of the recombinant protein and facilitates its detection, isolation, and purification.The recognized HA epitope represents the amino acid sequence YPYDVPDYA.|
|HAT Tag||KDHLIHNVHKEFHAHAHNK||Purif||The HAT (histidine affinity tag) epitope is a naturally-occurring sequence of non-adjacent histidine residues that has a lower overall charge than tags with consecutive His residues|
|His Tag||HHHHHH||SDS, WB, Crys, Func||Polyhistidine tags are useful for the detection and analysis of expressed recombinant proteins using immunoblotting, immunoprecipitation and immunostaining techniques.|
|Myc Tag||EQKLISEEDL||FACS, IAC, IA, IHC, WB||The c-Myc protein is a transcription factor, encoded by the c-Myc gene on human chromosome 8q24. c-Myc is commonly activated in a variety of tumor cells and plays an important role in cellular proliferation, differentiation, apoptosis, and cell cycle progression. A synthetic peptide corresponding to residues 410-419 of the human p62 c-myc protein conjugated to KLH is used as immunogen. c-Myc-tag provides a method of localizing gene products in a variety of cell types, to study the topology of proteins and protein complexes and of identifying associated proteins. It is advisable not to fuse the tag directly behind the signal peptide of a secretory protein, since it can interfere with translocation into the secretory pathway.|
|Rho Tag||MNGTEGPNFYVPFNSNKTGVV||Func, WB, IP, AbP||The Rho-tag is an epitope tag consisting of a twenty-residue peptide, MNGTEGPNFYVPFSNKTGVV, which is derived from the first 20 aa of bovine rhodopsin. Rho-tag can be used to boost expression levels of some eukaryotic membrane proteins or for their highly specific purification.|
|SNAP-Tag®||Fusionprotein (20 kDa)||SNAP- and CLIP-tag protein labeling systems® enable the specific, covalent attachment of virtually any molecule to a protein of interest. There are two steps to using this system: cloning and expression of the protein of interest as a SNAP-tag® fusion, and labeling of the fusion with the SNAP-tag® substrate of choice. The SNAP-tag® is a small protein based on human O6-alkylguanine-DNA-alkyltransferase (hAGT), a DNA repair protein. SNAP-tag® substrates are dyes, fluorophores, biotin, or beads conjugated to guanine or chloropyrimidine leaving groups via a benzyl linker. In the labeling reaction, the substituted benzyl group of the substrate is covalently attached to the SNAP-tag®. CLIP-tag™ is a modified version of SNAP-tag®, engineered to react with benzylcytosine rather than benzylguanine derivatives. When used in conjunction with SNAP-tag®, CLIP-tag™ enables the orthogonal and complementary labeling of two proteins simultaneously in the same cells.|
|Strep Tag II®||SA-WSHPQFEK||WB, IF, IP||The Strep-tag system® is a method which allows the purification and detection of proteins by affinity chromatography. The Strep-tag® is a synthetic peptide consisting of eight amino acids (Trp-Ser-His-Pro-Gln-Phe-Glu-Lys). This peptide sequence exhibits intrinsic affinity towards Strep-Tactin®, a specifically engineered streptavidin and can be N- or C- terminally fused to recombinant proteins. By exploiting the highly specific interaction, Strep-tagged proteins can be isolated in one step from crude cell lysates. Because the Strep-tag® elutes under gentle, physiological conditions it is especially suited for generation of functional proteins.|
|Twin-Strep Tag®||SA- WSHPQFEK (GGGS)2GGSA WSHPQFEK||WB||A tandemStrep-tag® formerly known as One-STrEP-tag® for a mild and rapid purification of intact protein complexes on immobilized Strep-Tactin®.|
|T7 Tag||MASMTGGQQMG||WB, SDS, IP, MS||The T7 epitope tag is commonly engineered onto the N- or C- terminus of a protein of interest so that the tagged protein can be analyzed and visualized using immunochemical methods. The recognized T7 epitope represents the amino acid sequence MASMTGGQQMG derived from the T7 major capsid protein of the T7 gene.|
|Trx Tag||Fusionprotein||SDS, WB, MS, Func||The Thioredoxin (TRX) tag is used predominantly to increase the solubility and thermal stability of proteins expressed in bacterial systems, where it also assists in the refolding of proteins requiring a reducing environment. It also provides a useful epitope tag, and has a molecular weight of approximately 12 kDa.|
|V5 Tag||GKPIPNPLLGLDST or IPNPLLGLD||WB, IF, IP||small epitope (Pk) present on the P and V proteins of the paramyxovirus of simian virus 5 (SV5) The V5 epitope tag is commonly engineered onto the N- or C- terminus of a protein of interest so that the tagged protein can be analyzed and visualized using immunochemical methods. The recognized V5 epitope represents amino acid residues 95-108, GKPIPNPLLGLDST, of RNA polymerase alpha subunit of simian virus 5.|
Abbreviations: Affinity Purification (AP), Antibody Production (AbP), Flow Cytometry (FACS), Functional Studies (Func), Chrystallization (Chrys), Detection (D), Immunocytochemistry (ICC), Immunoflorescence (IF), Immuno- histochemistry (IHC), Immunoprecipitation (IP), Immunoyassay (IA), Mass Spectronomy (MS), Protein Interaction (PI), Proximity Ligation Assay (PLA), Purification (Purif), Separation (Sep), SDS-PAGE (SDS), Western Blotting (WB)
What is a conjugate good for?
Conjugates can serve several purposes. In combination with a protein or peptide they are mainly used separate but also for the detection of the protein used or to distinguish between native and enhanced expression. Antibody conjugates mainly help in detection of the antibody and its binding partners but can als be used for separation. In immunization experiments conjugates (espcially KHL) are used extensively as a carrier protein in the production of antibodies. Low molecular weight haptens such as peptides, small proteins and drug molecules are generally not immunogenic and require the aid of a carrier protein in order to stimulate an immune response.