Antibody labeling is a procedure that has been with us for at least 6 decades now. The use of these labels has greatly helped increase the accuracy of the chemical procedures. Using the labels makes it easy to identify the antibodies and to isolate them for other experimental procedures. Substances that have the ability to fluoresce are typically chosen. These substances 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 other form of labelling is known as in vivo or metabolic conjugation. This type takes place in the body. Typically nucleic acids or amino acids are cultured for a couple of weeks to allow for the label to get attached to the proteins. As the labelling process takes place, RNA and DNA molecules undergo replication. Once the proteins have been identified, they are isolated, purified and used for other reactions.
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.
There are many types of enzymes that make use of active site probes. They include, among others, kinases, phosphatases, serine hydrolases, metalloproteases and the cytochrome p450 group of enzymes. The probes are used to assess the ability of molecules to cause inhibition as well as to quantify the activity of individual enzymes. The activity is an estimate of the potency. Many enzymes are themselves labels for many proteins. Commonly used enzymatic labels include glucose oxidase, alkaline phosphatase, horseradish peroxidase and so on.
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 other form of labelling is known as in vivo or metabolic conjugation. This type takes place in the body. Typically nucleic acids or amino acids are cultured for a couple of weeks to allow for the label to get attached to the proteins. As the labelling process takes place, RNA and DNA molecules undergo replication. Once the proteins have been identified, they are isolated, purified and used for other reactions.
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.
There are many types of enzymes that make use of active site probes. They include, among others, kinases, phosphatases, serine hydrolases, metalloproteases and the cytochrome p450 group of enzymes. The probes are used to assess the ability of molecules to cause inhibition as well as to quantify the activity of individual enzymes. The activity is an estimate of the potency. Many enzymes are themselves labels for many proteins. Commonly used enzymatic labels include glucose oxidase, alkaline phosphatase, horseradish peroxidase and so on.
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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