Multiple labeling

Craig, D.B., Dovichi, NJ. (1998). Multiple labeling of proteins Anal. Chem. 70, 2493-2494. 

Neher, R. and Neher, E. (2004). Optimizing imaging parameters for the separation of multiple labels in a fluorescence image. J. Microsc. 213, 46-62. 

Lissamine rhodamine B sulfonyl chloride has been used in numerous applications, including multiple-labeling techniques in microscopy (Wessendorf, 1990), for confocal microscopy... 

There are mainly three types of transducers used in immunosensors electrochemical, optical, and microgravimetric transducers. The immunosensors may operate either as direct immunosensors or as indirect ones. For direct immunosensors, the transducers directly detect the physical or chemical effects resulting from the immunocomplex formation at the interfaces, with no additional labels used. The direct immunosensors detect the analytes in real time. For indirect immunosensors, one or multiple labeled bio-reagents are commonly used during the detection processes, and the transducers should detect the signals from the labels. These indirect detections used to need several washing and separation steps and are sometimes called immunoassays. Compared with the direct immunosensors, the indirect immunosensors may have higher sensitivity and better ability to defend interference from non-specific adsorption. 

Notes In general, both secondary antibodies should come from the same host species for double/multiple labeling. 

Labeling with fluorescent phalloidins may be combined with immunostaining. In this case, the phalloidin-staining solution can be applied in a mixture with fluores-cently labeled secondary antibodies. Combination of immunostaining with fluorescent phalloidins and fluorescently counterstained nuclei are extremely useful in multiple labeling strategies to locate antigens of interest with specific components of the cell. 

The extent to which multiple fluorescence signals can be distinguished is demonstrated by resolution of five separate fluorochromes within individual, living 3T3 cells (24). None of the fluorochromes employed in this work was conjugated to an antibody, but the potential for extensive multiple labeling with a suitable set of fluorochromes and an appropriately equipped microscope system is clear. 

Slot, J. W. and Geuze, H. J. (1985) A new method of preparing gold probes for multiple-labeling cytochemistry. Eur. J. Cell Biol. 38, 87-93. 

Ontologies could go some way towards solving this problem since they assist in the specification of parthood relationships (e.g., between a single concept and multiple labels). 

If spots of multiple labeled amino acids (e.g., lysine, cysteine, histidine, tyrosine) are found, check whether the intensity of the multiple labeled derivatives is in the same order as the mono-labeled derivative. Only in that case could the determined amino acid be N-terminal. 

It can be prepared in several different sizes for use in multiple labeling experiments and quantitation, and... 

Rapid simple procedure. Multiple labeling is possible using different filter combinations. Confocal microscopy may be used. 

Reasonably rapid. Bright signal against a dark background is very sensitive. Many primary antibodies may be incorporated into the common procedure. Multiple labeling is possible using different filter combinations. Confocal microscopy may be used. 

The labeling must be specific, that is, directed to a specific site in the RNA to yield meaningful results pertaining to specific nucleotides. This is commonly referred to as site-directed spin-labeling (SDSL) (Altenbach et ah, 1989 Barhate et ah, 2007 Edwards et ah, 2001 Kim et ah, 2004 Qin et ah, 2001, 2003 Schiemann et ah, 2004). Therefore, incorporation of multiple labels through enzymatic RNA synthesis (e.g., triphosphate polymerization with polymerases Keyes et ah, 1997) is of limited value. Instead, labels are generally introduced chemically, either during chemical synthesis of the nucleic acid or by postsynthetic modification of the polymer. 

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