Peptides are short chains of amino acids linked together by peptide bonds. The peptide bonds are formed when the carboxyl group of one amino acid reacts with the amino group of another. Depending on how many amino acids there are in the chain the peptides may be called something different. For example, if the chain is two amino acids long, this is referred to as a dipeptide, and a chain of three amino acids is called a tripeptide. When this chain is more than 10 amino acids long, this will then be referred to as a polypeptide. Polypeptides are long, continuous and unbranched polypeptide chains.
Proteins are constructed of one or more polypeptide chains. Each protein consists of a specific sequence of amino acids that gives the primary structure of the protein. The secondary structure of the protein is formed by the formation of hydrogen bonds between polypeptide chains. Two common examples of secondary structures are the alpha helices and the beta pleated sheets. The tertiary 3-dimensional structure is then created by protein folding resulting from bond formation, including ionic bonds and disulfide bridges, and hydrophobic and hydrophilic interactions. The tertiary structure controls the basic function of a protein.
A conjugated protein is a compound made up of the protein or peptide of interest, and a molecule that allows for protein labelling and detection. Two common conjugations are bovine serum albumin (BSA) and horseradish peroxidase (HRP) Conjugated proteins and peptides are listed, with the conjugation included in the product name in brackets. For example, Hepcidin protein conjugated to BSA will be listed as ‘Hepcidin Protein (BSA)’.
A recombinant protein is a protein encoded by recombinant DNA. These proteins are genetically engineered and produced in bacterial, mammalian or yeast cells in order to make proteins in large quantities. Recombinant DNA molecules are made up of two or more segments of DNA joined together in a cloning vector, such as a bacterial plasmid. These cloning vectors are then used to transport the recombinant DNA into a host organism, such as E.coli. In the host, the recombinant DNA is replicated and protein-coding genes expressed. This allows for the production of known human and mammalian proteins in large quantities. In addition, recombination methods are good for creating new protein encoded by a novel combination of genes.
Blocking peptides are used to bind specifically to the target antibody and block antibody binding. The blocking peptide resembles the target epitope of the antibody and thus will bind to the target antibody to stop the antibody from binding to this epitope. This prevents non-specific antibody binding in order to give more accurate data and by decreasing background signal. The antibody specificity can then be assessed by comparing the strength of the signal between the unblocked antibody (control) and blocked antibody If this is assessed by Western blotting, the blocked antibody should display no signal.
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