Gold-conjugation

The amount of protein added at the stabilization point plus 10 percent should be used to produce the final protein-gold conjugate. 

Table 1.1 Binding profiles of protein A, protein G and protein A/G. Adapted from http // www.2spi.com/catalog/chem/gold conjugate.html)...

Transfer the grid to a droplet of gold conjugate diluted 1 10 to 1 100 (or greater) in PBS or Tris-buffered saline. Incubate for 1 h. 

Transfer the grid to a series of droplets of distilled water (5x2 min) to wash away unbound gold conjugates. 

Ready-to-use blocking solutions containing normal inactivated sera or BSA for use with colloidal gold conjugates are available from Aurion (http //www. aurion.nl/). When labeling with protein A and protein G conjugates, this step can be omitted. 

Colloidal gold A suspension (or colloid) of submicrometer-sized particles of gold in a fluid, usually water. A colloidal gold conjugate consists of gold particles coated with a selected protein or macromolecule, such as an antibody, protein A or protein G. Because of their high electron density, the gold particles are visible in the electron microscope without further treatment. 

Incubate slides for 30 min at room temperature in colloidal gold conjugate diluted in TBS plus 1% BSA. 

Colloidal gold conjugated with streptavidin (see Note 3). 

Add ligand conjugated to the colloidal gold, i.e., colloidal gold conjugated with antibody to a specific receptor. 

Colloidal gold-antibody conjugates are also widely used, especially in indirect immunocytochemical methods. However, the stability of these gold conjugates is less than that for protein A, and depending on the antibody, the affinity may be less. Colloidal gold conjugated to avidin (see... 

Incubate grids for 30 min at room temperature in colloidal gold conjugated with protein A, protein G, immunoglobulin, or if a biotinylated secondary antibody was used, streptavidin (see Note 6). 

Figure 1. Single hypha of C. versicolor grown on malt agar, labelled with rabbit anti-laccase antibodies, localized with goat-antirabbit gold conjugate. The label is restricted to the hyphal wall, with little label in the cytoplasm. Magnification x 16,000.

Not only biotinylated proteins, but also nucleic acids or peptides synthesized in the presence of biotin derivatives or carbohydrate-biotin conjugates, are separated by immobilized avidin and streptavidin, respectively, or are identified by avidin-enzyme or avidin-gold conjugates. 

Fig. 6 Carbohydrates in hydrogenosomes. Monocercomonas sp. after the Thiery technique (a). The hydrogenosomal membranes are positive for carbohydrates, b T. foetus cryosection labeled with gold-conjugated WGA. Hydrogenosome (H) shows that the membrane lining the peripheral vesicle, but not other portions of the organelle, is labeled. Bars = 100 nm. (Fig. 6a from Diniz and Benchimol 1998 Fig. 6b from Benchimol et al. 1996a)...

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