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 two types of labelling known; in vitro and in vivo. The former occurs outside the body. It involves a chemical reaction between one amino acid and another that acts as a label. A chemical reaction forms a covalent bond between the two amino acids. For this reaction to take place there are several things that are needed: a polymerase, a labelled amino acid or nucleotide and an ATP molecule.
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.
It is important to remember that the process of conjugation may affect the avidity of antibodies. There may be an increase or a reduction in the intrinsic activity. It is important to know the extent to which the substrate will be affected even as the reaction takes place. There are a number of assays that can be performed so as to assess the extent to which the avidity is affected.
There is a specific ratio between the protein and the label that has to be maintained. Optimal ratios are required for coupling reactions between the protein and the label to take place. There is a limit to the number of labels that can be attached to any single protein molecule. If this number if exceeded, the florescence is affected and identification is hindered. In other words the reagent that is produced is less dim than that which is optimally coupled.
A common application of the procedure is the use of active site probes. The active site is the place on an enzyme that reacts with the substrate. A probe that has detectable tag can be designed to attach to the enzyme. Apart from the tag, the probe also has a spacer and an active site. Since the probes are electrophilic, they can easily form a covalent bond with nucleophilic residues 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 many other applications and the list continues to grow by the day. There are ongoing efforts aimed at streamlining the procedure and improving the efficiency. At the same time, many manufacturers of reagents have incorporated the labels in their products.
There are two types of labelling known; in vitro and in vivo. The former occurs outside the body. It involves a chemical reaction between one amino acid and another that acts as a label. A chemical reaction forms a covalent bond between the two amino acids. For this reaction to take place there are several things that are needed: a polymerase, a labelled amino acid or nucleotide and an ATP molecule.
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.
It is important to remember that the process of conjugation may affect the avidity of antibodies. There may be an increase or a reduction in the intrinsic activity. It is important to know the extent to which the substrate will be affected even as the reaction takes place. There are a number of assays that can be performed so as to assess the extent to which the avidity is affected.
There is a specific ratio between the protein and the label that has to be maintained. Optimal ratios are required for coupling reactions between the protein and the label to take place. There is a limit to the number of labels that can be attached to any single protein molecule. If this number if exceeded, the florescence is affected and identification is hindered. In other words the reagent that is produced is less dim than that which is optimally coupled.
A common application of the procedure is the use of active site probes. The active site is the place on an enzyme that reacts with the substrate. A probe that has detectable tag can be designed to attach to the enzyme. Apart from the tag, the probe also has a spacer and an active site. Since the probes are electrophilic, they can easily form a covalent bond with nucleophilic residues 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 many other applications and the list continues to grow by the day. There are ongoing efforts aimed at streamlining the procedure and improving the efficiency. At the same time, many manufacturers of reagents have incorporated the labels in their products.
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