Serum proteins labelled

Heeeman JI (1977) The properties of a rat serum protein labeled by the injection of sodium selenite. Biochim Biophys Acta 500 61-70. 

In one set of experiments a titration of compound is performed to assess its potency in vivo. HeLa cells are maintained in DMEM supplemented with 10% fetal bovine serum (FBS) at 37° in 5% C02. One day prior to labeling, the cells are seeded in 24-well plates at approximately 60,000 cells per well. The next day, cells are washed with warm (37°) PBS and the medium replaced with 250 41 of methionine-free DMEM containing 10% dialyzed serum (Invitrogen). After a 15-min incubation at 37°, different concentrations of compound are added to the cells (which can range from 1 nM to 50 fiM) and the incubation continued for another 45 min. Anisomycin is used as a positive control at a final concentration of 50 /iM. Fifty-five microcuries of 35S-methionine/cysteine [35S-methionine/cysteine express protein labeling mix (1175 Ci/mmol) (Per-kin-Elmer)] is added to each well (220 /(Ci/ml) and the incubation continued for another 15 min. 

When cells are suspended in a biological fluid or culture medium, both serum proteins and cells interact with the surface substrate. Serum protein adsorption behavior on SAMs has been examined with various analytical methods, including SPR [58-61], ellipsometry [13, 62, 63], and quartz QCM [64—66]. These methods allow in situ, highly sensitive detection of protein adsorption without any fluorescence or radioisotope labeling. SPR and QCM are compatible with SAMs that comprise alkanethiols. In our laboratory, we employed SPR to monitor protein adsorption on SAMs. 

In addition to the application of roughened Ag electrodes in SERS measurements, one should add that Geddes et al. [15] have described the use of such Ag electrodes in metal-enhanced fluorescence studies also. The constant current flowing between two silver electrodes in pure water facilitated the growth of fractal-like structures on the cathode. The electrode was coated with a monolayer of human serum albumin protein labeled with Indocyanine Green. It was observed that the fluorescence intensity on the roughened electrode increased approximately the 50-fold, compared to the unroughened electrode. 

Figure 1 Reference 2-D PAGE map of rat serum. Unfractionated rat serum (30 jxl) was analyzed by 2-D PAGE. The separation was based on a pH 4-7 linear IPG in the first dimension and 11-18% SDS-PAGE in the second dimension. Proteins were visualized by staining with colloidal Coomassie. Some of the major proteins of rat serum are labeled.

Post-separation labeling was also achieved for separation of four human serum proteins (IgG, transferrin, al-antitrypsin, and albumin) using 0.2 mM of TNS. This is a virtually non-fluorescent reagent which, upon non-covalent association with proteins, produces a fluorescent complex (A= 325 nm, A = 450... 

Hill, R.D. and Laing, R.R., Specific reaction of dansyl chloride with one lysine residue in rennin, Biochim. Biophys. Acta 132, 188-190, 1967 Chen, R.F., Huorescent protein-dye conjugates. I. Heterogeneity of sites on serum albumin labeled by dansyl chloride. Arch. Biochem. Biophys. 128, 163-175, 1968 Chen, R.F., Dansyl-labeled protein modified with dansyl chloride activity effects and fluorescence properties. Anal Biochem. 25, 412M16, 1968 Brown, C.S. and Cunningham, L.W., Reaction of reactive sulfydryl groups of creatine kinase with dansyl chloride. Biochemistry 9, 3878-3885, 1970 Hsieh, W.T. and Matthews, K.S., Lactose repressor protein modified with dansyl chloride activity effects and fluorescence properties. 

Likhtenshtein and colleagues (Belonogova et al., 1978, 1979 Likhten-shtein, 1976) carried out a series of measurements on the hydration dependence of the mobility of spin labels covalently bound to several proteins. The results were correlated with Mossbauer spectroscopic data obtained in parallel experiments. Spin-labeled human serum albumin and a-chymotrypsin showed a critical hydration level for onset of motion at relative humidity 0.8, equivalent to 0.2 h. The temperature dependence of the spin label spectrum showed a critical temperature of 230 K, below which motion was frozen. Serum albumin labeled at surface sites... 

Lee DS, Chung JK, Lee MC. Preparation of E-18-human serum albumin A simple and efficient protein labeling method with... 

Kindmark, C. and Thorell, J (1972) Quanutauve determination of individual serum proteins by radioelectroimmunoassay and use of l-labelled antibodies (application toC-reacuve protein). Scand J. Cbn. Lab. Invest 29(Suppl. 124), 49-53. 

Fig. 4. Testing of a conjugate prepared from purified antibodies to mouse immunoglobulin labeled with alkaline phosphatase. Ordinate conjugate dilution abscissa absorbance at 405 nm of samples diluted 1 4 after 15 min of incubation with substrate. — , Dose response in microtiter wells coated with mouse serum proteins O—O, dose response in uncoated wells.

The proportion of a Tc bone agent which is taken up by bone after intravenous injection will be affected by its binding to proteins in the blood serum. The proportions of the plasma Tc which were protein bound two hours after administration were found to be 16, 31, 54 and 85% with HEDP, MDP, PYP and tripolyphosphate respectively. In addition, the proportion of the administered dose of Tc which was taken up in bone was found to increase linearly with the fraction of Tc in the plasma which was not protein bound. Cox has reported a linear decrease in bone uptake with molecular weight for this series of bone agents, an observation which appears to reflect differences in their plasma protein binding behaviour. Thus the blood serum proteins may compete with bone for the Tc complexes and reduce the bone labelling efficiency of the radiopharmaceutical. The "Tc-diphosphonate formulations showed better performance in this respect than "Tc-PYP or Tc-tripolyphosphate. 

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