Non-specific labeling

Protection should be immediately obvious when the results of a labeling experiment are examined by gel electrophoresis. A particularly clear-cut example is reproduced in Fig. 4.6 (Pomerantz et al., 1975 see also Fig. 4.4). Another instructive example can be seen in the work of Nordeen et al. (1981) in which specific labeling of glucocorticoid receptors was distinguished from a strongly but non-specifically labeled component of cell cytosol. 

If the photoaffinity reagent has been designed well and there are a measurable number of tight binding sites for it, the protection experiment will usually succeed. Occasionally it will not for example, four polypeptides of sarcoma cells were labeled with low concentrations of 8-N3-CAMP but only three of the sites could be protected by cAMP. Presumably, the fourth site is not part of a cAMP binding protein but a different nucleotide binding site that just happens to have affinity for the reagent but not for cAMP. 

Another test for specific labeling is to determine whether the ligand binding site is blocked. But, as 1 pointed out in the discussion of photoinactivation experiments, there are several other possible causes of apparent binding site occupation besides the covalent attachment of a ligand. It has also been noted that specific labeling as defined by a protection experiment may not always yield a blocked receptor when the photoaffinity label is a macromolecule. When sodium channels in tissue culture cells were labeled 

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Nitroaryl compounds including amino acid and peptide derivatives (Escher and Schwyzer, 1974), chloramphenicol (Sonenberg et al., 1974 which gave much non-specific labeling), and flunitrazepam (Mohler et al., 1980) have been used to photolabel receptors and they probably react via triplet... 

Fig. 4.4. Photoaffinity labeling of a benzodiazepine receptor. SDS-polyacrylamide gel electrophoresis of purified synaptic membranes after photoaffinity labeling with [3H]fluni-trazepam (3 nM). Right distribution of radioactivity in the gel. The hatched area is the label distribution when diazepam (10 pM) was present during photolysis (non-specific labeling). Left Coomassie blue staining pattern after irradiation (with and without diazepam present).

As no systematic study has been made of the concentration of a thiol required to scavenge various reactive species a study of the concentration dependence should be made in each case to ensure that maximal prevention of non-specific labeling is obtained. It should not be assumed that the efficacy of a thiol scavenger will be independent of pH as thiols have pKvalues of 9. 

Non-specific labeling prevented by anaerobic conditions. Azidonitro phenyl analog gave much non-specific labeling (Kac-zorowski et al., 1980). 

Banters et al. (2003) demonstrated Cryptococcus neoformans in cerebrospinal fluid (CSF) and serum. Their 30-min procedure was based on the non-specific labelling with ChemChrome V3 in combination with a second analysis using immunofluorescence. To that end, cells were labelled with a specific primary antibody against a capsular polysaccharide and a secondary antibody conjugated with FITC. 

Non-specific labeling - this condition exists when it is known that some atom(s) in the structure are labeled but the specific locations are unknown. In this case, the... 

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