Biological research often requires the use of molecular labels that are covalently attached to a protein of interest to facilitate detection or purification of the labeled protein and/or its binding partners. Labeling strategies result in the covalent attachment of different molecules, including biotin, reporter enzymes, fluorophores, and radioactive isotopes, to the target protein or nucleotide sequence. While multiple types of labels are available, their varied uses are preferable for specific applications. Therefore, the type of label and the labeling strategy used must be carefully considered and tailored for each application.
Biotin is a useful label for protein detection, purification and immobilization because of its extraordinarily strong binding to avidin, streptavidin or Thermo Scientific NeutrAvidin Protein. Indeed, this interaction is one of the strongest noncovalent interactions between a protein and ligand. Additionally, biotin (244.3 Da) is considerably smaller than enzyme labels and is therefore less likely to interfere with normal protein function. Together, these features make avidin–biotin strategies ideal for many detection and immobilization applications. However, depending on the nature of the application, the very strong binding interaction can be problematic. In those situations, certain variants of avidin or derivatives of biotin are available, which allow soft-release (elution) binding or cleavable (reversible) labeling.
Biotinylation is the process of labeling proteins or nucleotides with biotin molecules and can be performed by enzymatic and chemical means. Chemical methods of biotinylation are most commonly used, and the biotinylation reagents used for this type of labeling share several basic features. They are composed of the biotinyl group, a spacer arm and a reactive group that is responsible for attachment to target functional groups on proteins. Variations in these three features account for the many varieties of available reagents and provide the specific properties needed for particular applications.
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Spacer arms link the biotin molecule to a reactive group that interacts with certain functional groups on the amino acids of the target protein. Besides connecting biotin to a chemical group that mediates protein attachment, spacer arms can influence biotinylation and protein detection in three ways. First as indicated in the following diagram, these spacer arms vary by length, which can affect the availability of the attached biotin for binding to avidin, streptavidin or NeutrAvidin.
Examples of variable spacer arm lengths. Chemical groups (black) modify the distance between the reactive moiety (red) and biotin (blue) to regulate the length of the spacer arm. The reagents shown are (A) Thermo Scientific EZ-Link NHS-Biotin, (B) EZ-Link NHS-LC-Biotin and (C) EZ-Link Sulfo-NHS-LC-LC-Biotin.
Second, the solubility of a biotinylation reagent is an important factor that can influence its ability to biotinylate proteins that are located in membrane-bound compartments or alter the solubility of the labeled target protein. For example, a spacer arm consisting of poly(ethylene) glycol (PEG) repeats will increase or preserve the solubility of labeled proteins.
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Poly(ethylene glycol) increases the solubility of biotinylation reagents. A four-ethylene glycol chain (PEG4) was conjugated onto the spacer arm between the reactive moiety (red) and biotin (blue). The reagent shown is Thermo Scientific EZ-Link NHS-PEG4-Biotin.
In contrast, long hydrophobic spacer arms can render a labeled target protein less soluble but are ideal when performing labeling reaction in hydrophobic organic solvents such as dimethylsulfoxide (DMSO), which is often required when making modifying hydrophobic peptides. Third, spacer arms may contain a cleavable region (e.g., a reducible disulfide bond) that mediates separation of the biotin label from the protein to allow purification without harsh denaturants.
A wide range of biotinylation reagents with different reactive groups are commercially available. Common reactive groups and their respective targets on proteins include:
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Learn how to optimize your bioconjugation strategies with our updated Bioconjugation and crosslinking technical handbook. This easy-to-use guide overviews our portfolio of reagents for bioconjugation, crosslinking, biotinylation, and modification of proteins and peptides.
Active site probes are a class of chemical labeling reagents whose reactive groups are designed to specifically bind (label) particular enzyme active sites. Similar to traditional chemical labeling probes, active site probes contain a detectable tag (biotin/dye), a spacer arm and a reactive group that is responsible for attachment to the active site of the target class of enzymes. Active site reactive groups are typically electrophilic compounds which covalently link to nucleophilic residues found in enzyme active sites. In cases where the active site reactive group does not covalently bond to the target enzyme, photoreactive groups are incorporated into the linker region to facilitate attachment following specific binding. These probes can be used to selectively enrich, identify and profile target enzyme classes across samples or assess the specificity and affinity of enzyme inhibitors.
Active site probes have been developed to label different specific enzyme classes such as kinases, phosphatases, GTPases, serine hydrolases, cysteine proteases, metalloproteases and cytochrome p450 enzymes. All active site probes can be used to determine inhibition of enzymes by small molecules, and some probes also preferentially react with only active enzymes, allowing for activity-based proteomic profiling (ABPP). ABPP is a powerful method to monitor protein activity versus traditional protein or RNA expression profiling techniques that only measure abundance. The follow image and table illustrates processes involved in the detection of active serine hydrolase enzymes, and provides a list of serine hydrolases identified by mass spectrometry, respectively.
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Mechanism and chemical structures of active site probes for serine hydrolases. (A) Fluorophosphonate probes (FP) covalently and specifically attach to the active site serine of active serine hydrolases and proteases. (B) Structures of the azido, desthiobiotin and fluorescently-tagged fluorophosphonate probes for labeling, affinity enrichment or fluorescence detection of active serine hydrolase enzymes.
Serine hydrolases identified by mass spectrometry with ActivX fluorophosphonate (FP) probes. Number of serine hydrolase family members from mouse brain and liver tissue extracts identified by mass spectrometry after labeling and enrichment using the desthiobiotin-FP probe.
Bioconjugate Techniques, 3rd Edition(2013) by Greg T. Hermanson is a major update to a book that is widely recognized as the definitive reference guide in the field of bioconjugation.
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Bioconjugate Techniques is a complete textbook and protocols-manual for life scientists wishing to learn and master biomolecular crosslinking, labeling and immobilization techniques that form the basis of many laboratory applications. The book is also an exhaustive and robust reference for researchers looking to develop novel conjugation strategies for entirely new applications.It also contains an extensive introduction to the field of bioconjugation, which covers all the major applications of the technology used in diverse scientific disciplines, as well as tips for designing the optimal bioconjugate for any purpose.
Certain enzymes have properties that enable them to function as highly sensitive probes with a long shelf life and versatility for the detection of proteins in tissues, whole cells or lysates. Enzyme labels are considerably larger than biotin and require the addition of a substrate to generate a chromogenic, chemiluminescent or fluorescent signal that can be detected by different approaches. Enzyme labels are widely used because of their multiple types of signal output, signal amplification and the wide selection of enzyme-labeled products, especially antibodies.
Enzymes commonly used as labels include horseradish peroxidase (HRP), alkaline phosphatase (AP), glucose oxidase and β-galactosidase, and specific substrates are available for each enzyme. Indeed, multiple commercial substrates are available for HRP and AP that generate colorimetric, chemiluminescent or fluorescent signal outputs. To generate the following data, an anti-mouse IgG-HRP conjugated secondary antibody was used to perform immunohistochemistry (IHC).
Compartment Specific Labeling Of Bacterial Periplasmic Proteins By Peroxidase Mediated Biotinylation
Detection of p21 in human lung colon carcinoma by IHC. IHC staining for p21 in a formalin-fixed paraffin-embedded (FFPE) section of human colon carcinoma using a monoclonal antibody as the primary antibody and an anti-mouse IgG-HRP conjugate as the secondary antibody. The brown precipitating HRP substrate DAB was used. Prior to staining, heat-induced epitope retrieval (HIER) was performed in 10 mM citrate buffer.
Enzyme probes can be conjugated to antibodies, streptavidin or other target proteins by multiple mechanisms, including glutaraldehyde, reductive amination following periodate oxidation of sugars to reactive aldehydes, or by using heterobifunctional crosslinkers such as Sulfo-SMCC.
Fluorescent reagents of many types continue to be developed to stain or chemically label proteins, nucleic acids and other biomolecules. When specific antibodies or other purified biomolecules are chemically labeled with fluorescent dyes, they become fluorescent probes for detection of target antigens or interaction partners in applications such as cell imaging, high-content analysis, flow cytometry, western blotting and ELISA.
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The following representative examples include immunohistochemistry (IHC) and immunocytochemistry (ICC) data generated using multiple fluorescently-labeled probes that allow researchers to identify a variety of structures within the tissue or cell, respectively.
Immunocytochemistry analysis of adiponectin in HeLa cells. This experiment was performed using an Invitrogen ABfinity adiponectin recombinant rabbit monoclonal antibody followed by detection using an Invitrogen Alexa Fluor 488–conjugated goat anti-rabbit secondary antibody (green) (A). Nuclei were stained using DAPI (B) and actin stained with Alexa Fluor 594 phalloidin (red) (C). Image D is a composite image showing subcellular localization in the perinuclear region.
Detection of cytokeratin 18 in human colon carcinoma tissue by IHC using immunofluorescence. The sections were incubated with a biotinylated anti–cytokeratin 18 antibody and then detected using an Invitrogen streptavidin–DyLight 633 conjugate (red fluorescence). Invitrogen Hoechst stainwas used to counterstain the cell nuclei (blue fluorescence).
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Fluorescent molecules, also called fluorophores or simply fluors, respond directly and distinctly to light and produce a detectable
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