The use of antibody labeling techniques has revolutionized the field of research. It has made it possible to correctly identify proteins and to isolate them from a large pool. Most reagents that are used in chemical reactions today have molecules that are labelled. In most of these conjugation procedures, the label that is used can easily fluoresce to make the identification process easy. Labels that have this property are known as fluorophores.
There are a number of types of labelling that exist. One of them is the in vitro type. In this type of reaction, an amino acid is conjugated to a protein label such that a covalent bond is formed. There are a number of requirements that are necessary for this process to take place. They include polymerases, ATP molecules and amino acids or nucleotides that have been labelled.
The second type is in vivo or metabolic labelling. Metabolic labelling is done inside the body (in vivo). It involves the conjugation of both amino acids and nucleic acids. This is achieved by placing the proteins in a culture media for a couple of weeks. While in the culture medium the DNA and RNA molecules undergo replication and are all labelled. The next step is to identify the proteins of interest, to purify them and to use them as is desired.
The process of conjugation is associated with an interference with the avidity of the labelled proteins. The avidity may be increased, reduced or may remain the same. The extent to which the protein is interfered with depends on the type of label that is used. There are several assays that can be performed so as to determine the residual intrinsic activity of the antibody after the conjugation.
For the process of conjugation to take place, the ratio between the protein to be labelled and that to act as the label must be maintained at a certain critical value. The number of labels that are attached to each molecule has to be carefully controlled. In the event that this value is exceeded, the florescence will be interfered with and the reagents will not function as expected. Using a higher concentration of the labels makes the reagents less dim.
One of the commonest applications of this technique is active site probes. Active probes refer to reagents that bind to specific enzyme sites. They have a detectable tag, a spacer arm and a reactive group that attaches to the desired site. The probes are electrophilic in nature and form covalent links with nucleophilic residues that are found in enzymes. For this reason, the probes are used in the identification of enzymes.
Active site probes are used for enzymes such as metalloproteases, serine hydrolases, kinases and phosphatases and the cytochrome p450 enzymes. The probes are commonly used to asses for the inhibition ability of some molecules. They are also used to assess the activity of specific enzymes. This corresponds to the potency of the enzymes. There are a number of enzymes that are known to act as labels for other proteins including alkaline phosphatase, horseradish peroxidase and glucose oxidase among others.
Antibody labeling has made a huge difference in chemistry. It is now possible to work with virtually any type of protein substrate. This has made research fairly easy. There are ongoing research studies aimed at making the use of the labels even more efficiency.
There are a number of types of labelling that exist. One of them is the in vitro type. In this type of reaction, an amino acid is conjugated to a protein label such that a covalent bond is formed. There are a number of requirements that are necessary for this process to take place. They include polymerases, ATP molecules and amino acids or nucleotides that have been labelled.
The second type is in vivo or metabolic labelling. Metabolic labelling is done inside the body (in vivo). It involves the conjugation of both amino acids and nucleic acids. This is achieved by placing the proteins in a culture media for a couple of weeks. While in the culture medium the DNA and RNA molecules undergo replication and are all labelled. The next step is to identify the proteins of interest, to purify them and to use them as is desired.
The process of conjugation is associated with an interference with the avidity of the labelled proteins. The avidity may be increased, reduced or may remain the same. The extent to which the protein is interfered with depends on the type of label that is used. There are several assays that can be performed so as to determine the residual intrinsic activity of the antibody after the conjugation.
For the process of conjugation to take place, the ratio between the protein to be labelled and that to act as the label must be maintained at a certain critical value. The number of labels that are attached to each molecule has to be carefully controlled. In the event that this value is exceeded, the florescence will be interfered with and the reagents will not function as expected. Using a higher concentration of the labels makes the reagents less dim.
One of the commonest applications of this technique is active site probes. Active probes refer to reagents that bind to specific enzyme sites. They have a detectable tag, a spacer arm and a reactive group that attaches to the desired site. The probes are electrophilic in nature and form covalent links with nucleophilic residues that are found in enzymes. For this reason, the probes are used in the identification of enzymes.
Active site probes are used for enzymes such as metalloproteases, serine hydrolases, kinases and phosphatases and the cytochrome p450 enzymes. The probes are commonly used to asses for the inhibition ability of some molecules. They are also used to assess the activity of specific enzymes. This corresponds to the potency of the enzymes. There are a number of enzymes that are known to act as labels for other proteins including alkaline phosphatase, horseradish peroxidase and glucose oxidase among others.
Antibody labeling has made a huge difference in chemistry. It is now possible to work with virtually any type of protein substrate. This has made research fairly easy. There are ongoing research studies aimed at making the use of the labels even more efficiency.
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