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34-532: A collagen hybridizing peptide ( CHP ) is a synthetic peptide sequence with typically 6 to 10 repeating units of the Gly-Xaa-Yaa amino acid triplet, which mimics the hallmark sequence of natural collagens . A CHP peptide usually possesses a high content of proline and hydroxyproline in the Xaa and Yaa positions, which confers it a strong propensity to form the collagen's unique triple helix conformation. In

68-415: A better approach would be secondary IF, which is considered to be more sensitive than DIF when compared to Secondary (Indirect) Immunofluorescence. Secondary (indirect) immunofluorescence (SIF) is similar to direct immunofluorescence, however the technique utilizes two types of antibodies whereas only one of them have a conjugated fluorophore. The antibody with the conjugated fluorophore is referred to as

102-547: A defined 3D epitope. In addition, antibodies that were reported to distinguish specific degraded collagen fragments can only recognize one or few collagen types. In contrast, CHPs, in principle, can bind to all types of denatured collagens. Fluorophore- or biotin-labeled CHPs are used as a staining agent for detecting collagen degradation and denaturation via immunofluorescence and immunohistochemistry applications. CHPs can stain frozen tissue sections , formalin-fixed paraffin embedded (FFPE) sections, as well as fresh tissues. CHP

136-399: A diverse set of chemical manipulations on the developing product. These peptides are often cyclic and can have highly complex cyclic structures, although linear nonribosomal peptides are also common. Since the system is closely related to the machinery for building fatty acids and polyketides , hybrid compounds are often found. The presence of oxazoles or thiazoles often indicates that

170-660: A fluorophore has a broad emission specter, that overlaps with the specter of another fluorophore, thus giving rise to false signals. Non-specific staining occurs when the antibody, containing the fluorophore, binds to unintended proteins because of sufficient similarity in the epitope. This can lead to false positives. The main improvements to immunofluorescence lie in the development of fluorophores and fluorescent microscopes. Fluorophores can be structurally modified to improve brightness and photostability, while preserving spectral properties and cell permeability. Super-resolution fluorescence microscopy methods can produce images with

204-466: A major role in elucidating the 3D structure of the collagen triple helix , its folding kinetics, and thermal stability as small triple helical models. CMPs, CLPs, and CHPs are all very similar in terms of their amino acid sequences but only when CMPs or CLPs are heated above their melting temperatures, do they exist in the dissociated, single-stranded state and can be considered as CHPs. Single-stranded CHPs bind to denatured collagen chains and gelatin in

238-779: A manner that is unique from other targeting mechanisms, in that they specifically recognize a unique structural motif ( collagen triple helix ) for folding and chain assembly, as opposed to specific epitopes binding that is seen for monoclonal antibodies (mAbs), for example. Due to their unique targeting mechanism, CHPs have a high binding specificity towards denatured collagen chains but have almost no affinity for intact (triple helical) collagen. CHPs can broadly target collagen chains that have been denatured by thermal, chemical, mechanical, or enzymatic processes, as well as multiple collagen types (e.g., Col I , II , IV ). Studies also showed CHPs and their fluorophore conjugates have superior stability in contact with serum. Controlled collagen turnover

272-418: A single antibody, conjugated to a fluorophore . The antibody recognizes the target molecule (antigen) and binds to a specific region, called the epitope . The attached fluorophore can be detected via fluorescent microscopy, which, depending on the type of fluorophore, will emit a specific wavelength of light once excited. The direct attachment of the fluorophore to the antibody reduces the number of steps in

306-424: A specific predefined wavelength using a fluorescence microscope . It is imperative that the binding of the fluorophore to the antibody itself, do not interfere with the immunological specificity of the antibody or the binding capacity of its antigen. Immunofluorescence is a widely used example of immunostaining (using antibodies to stain proteins) and is a specific example of immunohistochemistry (the use of

340-691: A targeting moiety, CHPs offer great potential in histopathology , diagnostics, and drug delivery for a wide range of diseases. Most methods for the evaluation of collagen denaturation in disease states are indirect, such as detecting matrix metalloproteinase (MMP) activity or quantifying collagen peptide fragments in urine, serum, or synovial fluid . Using conventional methods for directly targeting collagen, researchers have to relied on collagen binding peptides selected by phage display , derived from collagen binding proteins, or antibodies raised against collagens. Unfortunately, these compounds cannot target denatured collagens which are unstructured and do not present

374-476: Is applicable to tissue specimens from multiple species and a range of diseases, such as myocardial infarction , arthritis , nephritis , and fibrosis . CHPs can also be labelled with near-infrared fluorophores for in vivo fluorescent imaging. CHPs can be used for visualizing many different types of collagen bands in SDS-PAGE gels. Collagen is denatured by heating in the presence of SDS prior to loading

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408-412: Is called an epitope . Several antibodies can recognize the same epitope but differ in their binding affinity. The antibody with the higher affinity for a specific epitope will surpass antibodies with a lower affinity for the same epitope. By conjugating the antibody to a fluorophore , the position of the target biomolecule is visualized by exciting the fluorophore and measuring the emission of light in

442-486: Is crucial for embryonic development, organ morphogenesis, as well as tissue maintenance and repair. However, changes of collagen homeostasis are associated with numerous diseases and pathological conditions. Excessive collagen degradation may be associated with cancer metastasis , skin ageing , arthritis , and osteoporosis . CHPs can target tissues undergoing remodelling based on their ability to bind to degraded and unfolded collagen strands through triple helix formation. As

476-419: Is employed in foundational scientific investigations and clinical diagnostic endeavors, showcasing its multifaceted utility across diverse substrates, including tissue sections, cultured cell lines , or individual cells. Its usage includes analysis of the distribution of proteins , glycans , small biological and non-biological molecules, and visualization of structures such as intermediate-sized filaments. If

510-480: Is estimated that at least 10% of the pharmaceutical market is based on peptide products. The peptide families in this section are ribosomal peptides, usually with hormonal activity. All of these peptides are synthesized by cells as longer "propeptides" or "proproteins" and truncated prior to exiting the cell. They are released into the bloodstream where they perform their signaling functions. Several terms related to peptides have no strict length definitions, and there

544-491: Is often overlap in their usage: Peptides and proteins are often described by the number of amino acids in their chain, e.g. a protein with 158 amino acids may be described as a "158 amino-acid-long protein". Peptides of specific shorter lengths are named using IUPAC numerical multiplier prefixes: The same words are also used to describe a group of residues in a larger polypeptide ( e.g. , RGD motif ). (See Template:Leucine metabolism in humans – this diagram does not include

578-1587: Is released during formation of each amide bond. All peptides except cyclic peptides have an N-terminal (amine group) and C-terminal (carboxyl group) residue at the end of the peptide (as shown for the tetrapeptide in the image). There are numerous types of peptides that have been classified according to their sources and functions. According to the Handbook of Biologically Active Peptides , some groups of peptides include plant peptides, bacterial/ antibiotic peptides , fungal peptides, invertebrate peptides, amphibian/skin peptides, venom peptides, cancer/anticancer peptides, vaccine peptides, immune/inflammatory peptides, brain peptides, endocrine peptides , ingestive peptides, gastrointestinal peptides, cardiovascular peptides, renal peptides, respiratory peptides, opioid peptides , neurotrophic peptides, and blood–brain peptides. Some ribosomal peptides are subject to proteolysis . These function, typically in higher organisms, as hormones and signaling molecules. Some microbes produce peptides as antibiotics , such as microcins and bacteriocins . Peptides frequently have post-translational modifications such as phosphorylation , hydroxylation , sulfonation , palmitoylation , glycosylation, and disulfide formation. In general, peptides are linear, although lariat structures have been observed. More exotic manipulations do occur, such as racemization of L-amino acids to D-amino acids in platypus venom . Nonribosomal peptides are assembled by enzymes , not

612-512: Is the main component of the extracellular matrix (ECM). The collagen superfamily consists of 28 different types of collagen. Although the function and hierarchical structure of these collagens may vary, they all share the defining structural feature known as the triple helix, where three left handed polyproline II-type (PPII) helices assemble to form a right-handed supercoiled helical motif. Short synthetic peptides known as collagen mimetic peptides (CMPs) or collagen-like peptides (CLPs) have played

646-404: The antibody-antigen relationship in tissues). This technique primarily utilizes fluorophores to visualize the location of the antibodies, while others provoke a color change in the environment containing the antigen of interest or make use of a radioactive label. Immunofluorescent techniques that utilized labelled antibodies was conceptualized in the 1940s by Albert H. Coons . Immunofluorescence

680-506: The broad chemical classes of biological polymers and oligomers , alongside nucleic acids , oligosaccharides , polysaccharides , and others. Proteins consist of one or more polypeptides arranged in a biologically functional way, often bound to ligands such as coenzymes and cofactors , to another protein or other macromolecule such as DNA or RNA , or to complex macromolecular assemblies . Amino acids that have been incorporated into peptides are termed residues . A water molecule

714-489: The cell, as antibodies generally do not penetrate intact cellular or subcellular membranes in living cells, because they are large proteins. To visualize these structures, antigenic material must be fixed firmly on its natural localization inside the cell. To study structures within living cells, in combination with fluorescence, one can utilize recombinant proteins containing fluorescent protein domains, e.g., green fluorescent protein (GFP). The GFP-technique involves altering

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748-430: The compound was synthesized in this fashion. Peptones are derived from animal milk or meat digested by proteolysis . In addition to containing small peptides, the resulting material includes fats, metals, salts, vitamins, and many other biological compounds. Peptones are used in nutrient media for growing bacteria and fungi. Peptide fragments refer to fragments of proteins that are used to identify or quantify

782-451: The concentration of fluorophores, or opt for more robust fluorophores that exhibit resilience against photobleaching such as Alexa Fluors , Seta Fluors, or DyLight Fluors . Other problems that may arise when using immunofluorescence techniques include autofluorescence , spectral overlap and non-specific staining. Autofluorescence includes the natural fluorescence emitted from the sample tissue or cell itself. Spectral overlap happens when

816-473: The gel. The collagen bands are visualized through CHP-collagen hybridization when the gels are stained by fluorescently-labeled CHPs. Collagen offers mechanical strength in load bearing tissues in the body such as tendons, ligaments, and bone. As forces are applied to these tissues, the collagen triple helix can be damaged and unwind, and CHPs allow for molecular level detection of mechanical damage in such connective tissues. Peptide Peptides fall under

850-449: The genetic information of the cells. A significant problem with immunofluorescence is photobleaching , the fluorophores permanent loss of ability to emit light. To mitigate the risk of photobleaching one can employ different strategies. By reducing or limiting the intensity, or timespan of light exposure, the absorption-emission cycle of fluorescent light is decreased, thus preserving the fluorophores functionality. One can also increase

884-432: The pathway for β-leucine synthesis via leucine 2,3-aminomutase) Immunofluorescence Immunofluorescence (IF) is a light microscopy -based technique that allows detection and localization of a wide variety of target biomolecules within a cell or tissue at a quantitative level. The technique utilizes the binding specificity of antibodies and antigens . The specific region an antibody recognizes on an antigen

918-434: The ribosome. A common non-ribosomal peptide is glutathione , a component of the antioxidant defenses of most aerobic organisms. Other nonribosomal peptides are most common in unicellular organisms , plants , and fungi and are synthesized by modular enzyme complexes called nonribosomal peptide synthetases . These complexes are often laid out in a similar fashion, and they can contain many different modules to perform

952-498: The same primary antibody. The increased number of fluorophore molecules per antigen increases the amount of emitted light, and thus amplifies the signal. There are different methods for attaining a higher fluorophore-antigen ratio such as the Avidin-Biotin Complex (ABC method) and Labeled Streptavidin-Biotin (LSAB method). Immunofluorescence is only limited to fixed (i.e. dead) cells, when studying structures within

986-430: The sample preparation procedure, saving time and reducing non-specific background signal during analysis. This also limits the possibility of antibody cross-reactivity, and possible mistakes throughout the process. One disadvantage of DIF is the limited number of antibodies that can bind to the antigen. This limitation may reduce sensitivity to the technique. When the target protein is available in only small concentrations,

1020-462: The secondary antibody, while the unconjugated is referred to as the primary antibody. The principle of this technique is that the primary antibody specifically binds to the epitope on the target molecule, whereas the secondary antibody, with the conjugated fluorophore, recognizes and binds to the primary antibody.   This technique is considered to be more sensitive than primary immunofluorescence, because multiple secondary antibodies can bind to

1054-484: The single-stranded (monomeric) status, the peptide can recognize denatured collagen strands in tissues by forming a hybridized triple helix with the collagen strands. This occurs via the triple helical chain assembly and inter-chain hydrogen bonding, in a manner similar to primers binding to melted DNA strands during PCR . The binding does not depend on a specific sequence or epitope on collagen, enabling CHPs to target denatured collagen chains of different types. Collagen

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1088-562: The source protein. Often these are the products of enzymatic degradation performed in the laboratory on a controlled sample, but can also be forensic or paleontological samples that have been degraded by natural effects. Peptides can perform interactions with proteins and other macromolecules. They are responsible for numerous important functions in human cells, such as cell signaling, and act as immune modulators. Indeed, studies have reported that 15-40% of all protein-protein interactions in human cells are mediated by peptides. Additionally, it

1122-440: The topology of a cell membrane is undetermined, epitope insertion into proteins can be used in conjunction with immunofluorescence to determine structures within the cell membrane. Immunofluorescence (IF) can also be used as a “semi-quantitative” method to gain insight into the levels and localization patterns of DNA methylation. IF can additionally be used in combination with other, non-antibody methods of fluorescent staining, e.g.,

1156-733: The use of DAPI to label DNA . Examination of immunofluorescence specimens can be conducted utilizing various microscope configurations, including the epifluorescence microscope , confocal microscope , and widefield microscope. To perform immunofluorescence staining, a fluorophore must be conjugated (“tagged”) to an antibody. Staining procedures can be applied to both retained intracellular expressed antibodies, or to cell surface antigens on living cells. There are two general classes of immunofluorescence techniques: primary (direct) and secondary (indirect). The following descriptions will focus primarily on these classes in terms of conjugated antibodies. Primary (direct) immunofluorescence (DIF) uses

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