Labeling with colloidal gold

Mannans.—A j8-(l -> 3)-glucanase has been used to prepare protoplasts from C. utilis and S. cerevisiae. Scanning electron microscopy of prefixed protoplasts, using colloidal gold labelled with concanavalin A, showed that the D-mannan is distributed randomly at the surface of the protoplasts. 

Apply goat antimouse or antirabbit secondary antibody labeled either with colloidal gold or with horseradish peroxidase for 30 min (see Notes 5 and 11). Wash for 2 X 2 min in TBS containing 0.1% Tween-20. 

Tris(hydroxymethyl)methylamine (TRIS), sodium chloride, sodium citrate, ethylenediamine tetraacetic acid disodium salt (EDTA), lithium chloride, Tween 20, streptavidin 10 nm colloidal gold labelled, hydrochloric acid (37%), nitric acid, streptavidin-coated paramagnetic beads (MB) with a diameter of 2.8 pm, Dynabeads M-280 Streptavidin (Dynal Biotech, Oslo, Norway) biotinylated probe oligonucleotides which sequences are shown in Table 53.1. 

New techniques based on immuno-chromatography uses colloidal gold-labeled monoclonal antibodies to detect E. coli 0157 117. These antibodies form complexes with E. coli 0157 117 as the test sample migrates over a membrane. Positive reactions show a colored band that is compared to a reference scale provided by the manufacturer. This technique enables a quick detection and was employed in the ImmunoCard Stat E. coli 0157 117 assay (Meridian Diagnostics). Mackenzie et al. (2000) evaluated this commercial kit on stool samples. All the 14 culture samples positive for E. coli 0157 117 were detected in contrast to the 263 culture-negative specimens that were not. The authors therefore concluded that the test was highly sensitive and specific. 

Colloidal gold - labeling for postembedding electron microscopic immunocyto-chemistry uses a colloids of gold with sizes from 5 to 50 nm attached to label secondary antibodies or protein A. 

The first report of this type was by Zavala and Brandon [48] who sectioned root tips at low temperature in order to try and prevent the redistribution of hormones. Their tissue sections were then probed with dihydrozeatin antibodies labelled with rhodamine. For electron microscopy, samples were freeze-substituted in ethanol or acetone and then embedded in polymers prior to sectioning and probing with colloidal gold-labelled antibodies. Although apparent specific labelling was reported in this work, it has been criticised [147] because dihydrozeatin is a relatively rare cytokinin, that had not been... 

Wash with UHQ.water and silver enhance the colloidal gold label (see Protocol 10). 

Stain colloidal gold labelled sections with Toluidine blue, wash, and air dry. 

One particularly novel carrier was reported to consist of 50-70 nm colloidal gold particles of the type often used in cytochemical labeling techniques for microscopy (Pow and Crook, 1993) (Chapter 24). Adsorption of peptide antigens onto gold and subsequent injection of the complex into rabbits in an adjuvant mixture resulted in rapid production of antibody of extremely high titer. The resultant antibodies could be used in immunocytochemistry at dilutions from l-in-250,000 down to l-in-1,000,000, which is orders-of-magnitude beyond the dilutions typically used with lower-titer antibodies. 

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