Antibody labeling has been a great step in increasing the accuracy in chemical reactions. The process has made it possible to set up procedures that were previously considered impossible. This is because many protein substrates can be easily identified and isolated unlike in the past. The labels that are commonly used have the ability to fluoresce and 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.
One of the greatest causes for concern is the fact that the conjugation process may result in interference of the intrinsic activity of the antibody or protein. Depending on the type of label that is used the avidity may be affected to different extents or may even remain unchanged. There are a number of methods that can be employed to determine the residual activity after the combination between the protein and the label.
It is important to ensure that the ration between the labels and the substrate is maintained within the recommended value. An optimal combination ration ensures that all the substrates are labelled as the conjugation reaction takes place. When the number of labels for each molecule is too high, the fluorescence may not take place and this may pose a great challenge to the person performing the chemical reaction.
The use of this procedure in active site probes is one of the commonest applications. The probes are designed to bind to an active site found on an enzyme. They are attached to a detectable tag, a reactive group and a spacer arm. The probes are electrophilic and easily react with nucleophilic residues on enzymes to form covalent links. The probes are for these reasons, used to identify different types of enzymes.
There are many types of enzymes that make use of active site probes as labels including kinases, phosphatases, serine hydrolases, metalloproteases, and cytochrome enzymes and so on. Among the enzymes, the probes are very useful in assessing the degree of inhibition by various types of molecules. They also are used in assessing the intrinsic activity of individual enzymes. The enzymes may also act as labels for other types of proteins. Examples of proteins that act in this manner include horseradish peroxidase, glucose oxidase and alkaline peroxidase.
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.
One of the greatest causes for concern is the fact that the conjugation process may result in interference of the intrinsic activity of the antibody or protein. Depending on the type of label that is used the avidity may be affected to different extents or may even remain unchanged. There are a number of methods that can be employed to determine the residual activity after the combination between the protein and the label.
It is important to ensure that the ration between the labels and the substrate is maintained within the recommended value. An optimal combination ration ensures that all the substrates are labelled as the conjugation reaction takes place. When the number of labels for each molecule is too high, the fluorescence may not take place and this may pose a great challenge to the person performing the chemical reaction.
The use of this procedure in active site probes is one of the commonest applications. The probes are designed to bind to an active site found on an enzyme. They are attached to a detectable tag, a reactive group and a spacer arm. The probes are electrophilic and easily react with nucleophilic residues on enzymes to form covalent links. The probes are for these reasons, used to identify different types of enzymes.
There are many types of enzymes that make use of active site probes as labels including kinases, phosphatases, serine hydrolases, metalloproteases, and cytochrome enzymes and so on. Among the enzymes, the probes are very useful in assessing the degree of inhibition by various types of molecules. They also are used in assessing the intrinsic activity of individual enzymes. The enzymes may also act as labels for other types of proteins. Examples of proteins that act in this manner include horseradish peroxidase, glucose oxidase and alkaline peroxidase.
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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