Radiolabeling procedure

The following operations should be done using standard safety procedures for working with radioactive compounds. All steps involving SASD prior to initiation of the photoreaction should be done protected from light to avoid loss of phenyl azide activity. The radiolabeling procedure should be done quickly to prevent excessive loss of NHS ester activity due to hydrolysis. 

We chose to prepare 14C-methyl-K-casein (M-k-C) as a tracer because of the important role of K-casein in stabilizing casein micelles (8) and because K-casein is known to participate in heat-induced interactions with whey proteins, thereby influencing the heat stability of milk (9). The reductive methylation radiolabeling procedure used low concentrations of reagents (10) and resulted in M-k-C containing approximately 1 fiinol of 14C-methyl groups for every micromole of protein monomer (about 3 /xCi/mg). When tracer M-k-C was added to skim milk, and trichloroacetic acid was added to a concentration of 2%, about 1% of the radioactivity remained soluble. After clotting of the milk with excess... 

The radiolabelling procedures were standardized with the help of optimization studies of labelling parameters such as the peptide to radionuclide molar ratio, pH, temperature and time of reaction. 

Fig. 8.9. Human platelets after the radiolabeling procedure, examined by transmission electron microscopy. Almost no signs of platelet activation, manifested as pseudopodia formation. Perfusion fixation, critical point drying, x 40 500...

Mucin was extracted from bovine submaxi 11 ary glands and collagen was isolated from rat tail tendons. The experimental protocols describing extraction, isolation and the radiolabeling procedures of these proteins were reported in Refs.(8, 11). 

The trichothecene mycotoxins are cytotoxic to most eukaryotic cells.46 A number of cytotoxicity assays have been developed and include survival and cloning assays, measuring protein and deoxyribonucleic acid (DNA) synthesis by radiolabeling procedures, and a neutral red cell-viability assay. A minimum of 24 to 48 hours is required to measure the effects of the trichothecene mycotoxins on cell viability. 

The radiolabeling procedure does not alter the dose and particle size distribution of the aerosol in comparison to the unlabeled drug. 

Radiolabeling Procedure. [ C]-Formaldehyde labeling of siuface A was performed in a self-fabricated metal sample holder with four wells (diameter = 7 mm). Acetonitrile (50 fiL) containing 10.0 mM NaBHsCN and 2.0 mM [ C]-formaldehyde was iiyected into each well. After incubation for 4 h at 20 °C, the excess radioactive solution was removed and the exposed surface washed with CH3CN 10 times and water 10 times. The demounted samples were extensively washed again with water and then dried with N2. The same procedure was also applied to the control surfaces of Ti and acetylated A The radioactivity of each sample was measured by scintillation counting in 5 mL of scintillation fluid (1080 mL of toluene, 920 mL of Triton X-100, 5.4 g of 2,5-diphenyloxazole, 0.2 g of l,4-bis-2-(5-phenyloxazolyl) benzene, and 40 mL of acetic acid) on a Tri-Carb 2300TR liquid scintillation counter (Packard Instrument Co., USA). 

The new finding that the polyST complex was located on the cytoplasmic surface of the inner membrane required that either oligo- or polySia chains must traverse this membrane before being exported to the outer leaflet of the outer membrane. To study directly the translocation of polySia chains across the inner membrane, an in vivo radiolabeling procedure was developed (Troy et al., 1990a,c, 1991). In this method, spheroplasts were prepared from E. coli K1 cells unable to degrade Sia because of a mutation in Sia aldolase nanM), and from a translocation-... 

---