Enzymes
Enzymes offer the advantages of long shelf life, high sensitivity and the possibility of direct visualization. The disadvantages of enzymes as labels include multiple assay steps, some hazardous substrates and the possibility of interference from endogenous enzymes. Enzymes also may give poor resolution in cytochemistry. The use of enzyme labels is recommended for immunohistochemistry, immunoblots, and quantitative and qualitative immunoassays.
Using enzymes as labels offers several advantages over fluorescently labeled and radiolabeled substances. Enzyme immunoassay reagents are more stable and do not have the dangers associated with radioisotopic labels. In addition, enzyme assays can be at least as sensitive as radioimmunoassays. Many enzyme detection methods are visual or use a standard spectrophotometer, eliminating the need for expensive, sophisticated equipment.
Enzyme labels have additional advantages when used to detect cellular antigens to tissue structures. Antibodies (and especially their fragments) conjugated to small enzymes can readily cross cell membranes. It is then possible to localize and observe cellular antigens to tissue structures using light microscopy. In addition, tissue sections that have been developed with the appropriate substrate can be stable for years. This is in stark contrast to most immunofluorescent staining techniques, in which the fluorescent signal rapidly decreases upon exposure to light.
Selection of an enzyme and the appropriate substrate solution are dependent on the application involved (Table 1). Enzyme-labeled antibodies perform well in immunoblotting, histochemical staining and ELISA. They can provide an instant visual result and good sensitivity; however, the rate of the enzyme reaction must be measured to obtain an accurate indication of the amount of bound enzyme, making them difficult to use in quantitative assays. In addition, enzyme-labeled reagents are not homogeneous. In immunohistochemistry, nonspecific staining can present a problem when using enzymes; additional problems include endogenous peroxidase or phosphatase activity. In these cases, a different enzyme may need to be selected.
The link provided also provides a comprehensive list of advantages/disadvantages. Hope this helps!!
Enzymes
Enzymes offer the advantages of long shelf life, high sensitivity and the possibility of direct visualization. The disadvantages of enzymes as labels include multiple assay steps, some hazardous substrates and the possibility of interference from endogenous enzymes. Enzymes also may give poor resolution in cytochemistry. The use of enzyme labels is recommended for immunohistochemistry, immunoblots, and quantitative and qualitative immunoassays.
Using enzymes as labels offers several advantages over fluorescently labeled and radiolabeled substances. Enzyme immunoassay reagents are more stable and do not have the dangers associated with radioisotopic labels. In addition, enzyme assays can be at least as sensitive as radioimmunoassays. Many enzyme detection methods are visual or use a standard spectrophotometer, eliminating the need for expensive, sophisticated equipment.
Enzyme labels have additional advantages when used to detect cellular antigens to tissue structures. Antibodies (and especially their fragments) conjugated to small enzymes can readily cross cell membranes. It is then possible to localize and observe cellular antigens to tissue structures using light microscopy. In addition, tissue sections that have been developed with the appropriate substrate can be stable for years. This is in stark contrast to most immunofluorescent staining techniques, in which the fluorescent signal rapidly decreases upon exposure to light.
Selection of an enzyme and the appropriate substrate solution are dependent on the application involved (Table 1). Enzyme-labeled antibodies perform well in immunoblotting, histochemical staining and ELISA. They can provide an instant visual result and good sensitivity; however, the rate of the enzyme reaction must be measured to obtain an accurate indication of the amount of bound enzyme, making them difficult to use in quantitative assays. In addition, enzyme-labeled reagents are not homogeneous. In immunohistochemistry, nonspecific staining can present a problem when using enzymes; additional problems include endogenous peroxidase or phosphatase activity. In these cases, a different enzyme may need to be selected.
The link provided also provides a comprehensive list of advantages/disadvantages. Hope this helps!!
References :
http://www.piercenet.com/Objec.....7E413910AB
Because you ran out of the right reagent.
References :