Isothiocyanates Experimental Procedure

Isothiocyanates can be prepared from support-bound primary amines by treatment with thiophosgene [14] or synthetic analogs thereof (Entry 5, Table 14.2). In an alternative two-step procedure, the amine is first treated with CS2 and a tertiary amine to yield an ammonium dithiocarbamate, which is subsequently desulfurized with TsCl or a chloroformate (Entry 6, Table 14.2 Experimental Procedure 14.1). Highly reactive acyl isothiocyanates have been prepared from support-bound acyl chlorides and tetra-butylammonium thiocyanate (Entry 7, Table 14.2). These acyl isothiocyanates react with amines to give the corresponding 7V-acylthiourcas, which can be used to prepare guanidines on insoluble supports (Entry 6, Table 14.3). 

Experimental Procedure 14.1 Conversion of polystyrene-bound primary aliphatic amines into isothiocyanates [26]... 

BARS concentration by some 5-fold to 15-fold, based on the calculations that the intracellnlar concentration is around 20 //g/ml and on the assumption that 5-10% of the cell volume is injected. Prior to injection, the protein is mixed with 0.4 mg/ml fluorescein isothiocyanate (FITC)- or tetramethyl-rhodamine B isothiocyanate (TRITC)-labeled dextran (Molecnlar Probes) as a marker for the microinjected cells (Bonazzi et al., 2005). To give an example, in stndies of basolateral and apical transport (using the vesicular stomatitis virus glycoprotein and p75, respectively), the proteins were microinjected 1 h after the beginning of the 20° incubation in the transport assay. After injection, the cells were allowed to recover for 1 h before proceeding with the experimental protocol (Bonazzi et al, 2005). The BARS (p50-2) and dynamin (DYN2) antibodies were injected at 4.5 mg/ ml, 3 h before farther experimental procedures. 

III.l Experimental Procedure The samples were made of variable amounts of hydrophobic colloidal silica (Aerosol R972 Degussa) mixed with an index matching solvent (ethanol + ethanolamine) containing the tracer molecule (fluorescein isothiocyanate. Molecular Probes). We have checked that tracer adsorption on the silica was negligible. Further details on the sample preparation can be found in reference [16]. 

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