Proteins radio-labelling

Slepnev et al. [313] presented a simple method for protein radio-labeling with Bolten-Hunter reagent (iV-hydroxy succinimide ester of 3-(p-hydroxy di-iodophenyl)) propionic acid) in AOT/octane-RMs using mouse IgG, human transferrin, protein A, and a2-interferon as labeling examples. The yield of radio-labeling in RMs was found to be higher than that achieved in homogeneous aqueous solution. 

Figure 2. Schematic illustration of protein radio-labelling and elastase disgestion.

Radioimmunoassay is a competitive protein binding assay which utilizes an antibody as the binding protein. This assay also employs a highly purified antigen which has been radio-labeled (tagged). 

L-type Ca channels are the only Ca channels that have been extensively characterized at the biochemical level. The success in characterizing L-type channels was largely due to their rich pharmacology, and in particular to the availability of radio-labelled DHPs. However, a second major contributor to this successful characterization came from the seminal findings of Glossmann and colleagues [45] and Fosset, Lazdunski and colleagues [43] who identified skeletal muscle transverse tubule membranes as a relatively rich source of this rare membrane protein. 

The result of a standard photoaffinity experiment can provide an output in three levels [8] as shown in Scheme 2. The photolytically induced targeted introduction of a (radio )label onto the target biopolymer helps to identify any specific binding proteins (first level of identification) responsible for a particular action in the signaling cascade or to identify the binding domain within the target protein (second level of identification) or allows one to identify the amino acid sequence of the binding protein (third level of identification). 

This assay method (RIPA) is used primarily in research. It is too technically demanding for routine use in clinical laboratories. HIV is cultured in radio-labeled cells, or viral proteins are directly labeled with a radioisotope. The virus is disrupted and then exposed to the test specimen. Specific antigen-antibody complexes are concentrated and isolated by immunoprecipitation. After exten-... 

DNA and/or protein vaccine entrapment in DRV liposomes is monitored by measuring the vaccine in the suspended pellet and combined supernatants. The most convenient way to monitor DNA entrapment is by using radio-labelled or DNA. For protein entrapment, the use of I-labelled protein tracer is recommended. If a radiolabel is not available or cannot be used, appropriate quantitative techniques should be employed. To determine DNA or protein by such techniques, a sample of the liposome suspension is mixed with Triton X-100 (up to 5% final concentration) or, preferably, with isopropanol (1 1 volume ratio) so as to liberate the entrapped materials. However, if Triton X-100 or the solubilized liposomal lipids interfere with the assay of the materials, liposomal lipids or the DNA must be extracted using appropriate techniques (6). Entrapment values for protein and DNA, whether alone or coentrapped, range between about 20% to 80% (protein) and 30%i to 100%i (DNA) of the initial material depending on the DNA or protein used and, in the case of DNA, the presence or absence of cationic charge. Values are highest for DNA when it is entrapped into cationic DRV (typical values in Table 1). 

Studies of canavanine interaction with the tobacco hornworm and J-. miTior also revealed the marked ability of canavanine.to inhibit whole organism incorporation of [ Hjthymidine and uridine into trichloroacetic acid-precipitated materials. When canavanine is provided simultaneously with the appropriate radio-labeled precursor, ample evidence for curtailed nucleic acid metabolism emerges but protein synthesis is unaffected (Table I, exp. I). In experiment II, canavanine is allowed to assimilate... 

Figure 7.18 (A/B) Relationship between hepatic glutathione, covalent binding of radio-labeled paracetamol to hepatic protein, and urinary excretion of paracetamol mercapturic acid after different doses of paracetamol. Source. From Ref. 9.

Figure 7.38 Dose-response curves for lethality (%, o), oedemagenesis (wet weight dry weight ratios, ) and pulmonary covalent binding (nmol.mg-1 protein, A) of radio-labelled 4-ipomeanol given i.p. to the rat. Source From Ref. 15.

Immediately following inhalation of labelled methyl chloride by rats, up to 20% of the label was incorporated into tissue macromolecules. After 6 h, the total level of non-volatile label was highest in liver and kidney and lower in testes. Within 24 h, about 64% of the label was exhaled, 32% found in urine and about 4% in faeces. About 50% of the radio-label was expired as [ C]CO2. Following oral administration, radioactivity in hepatic proteins was associated with methionine and serine. 

Chloramphenicol acetyl transferase (CAT). This bacterial enzyme was the first reporter protein used for studying the transcriptional activity of eukaryotic regulatory sequences (Gorman et al., 1982). CAT inactivates chloramphenicol, an inhibitor of prokaryotic protein synthesis, by converting it to the mono- or di-acetylated species. Measurement of CAT activity requires a 14C-radiolabeled chloramphenicol or acetyl-CoA and, therefore, an additional step is neccessary to separate the radio-labeled reactant from the product. Novel detection methods based on fluorescent substrates or ELISA assays, which do not use radiolabeled reagents, have been described more recently (Bullock and Gorman, 2000). 

The results on the hydrolysis of partially methylated /3-casein by plasmin indicate that proteins radiomethylated to a low level can serve as substrates for trypsin-like enzymes and probably for proteinases in general. Because it is likely that methylation will interfere with enzymatic attack at lysine residues, the complete hydrolysis of /3-casein probably would not be possible. Studies on mastitic milk demonstrate the usefulness of 14C-methyl proteins for qualitative examination of protein hydrolysis in complex multiprotein systems where resolution and characterization of individual protein fragments is difficult. The requirements in such studies are the availability of pure samples of the proteins under investigation and a suitable technique for separating the radio-labeled protein from hydrolytic products. 

Fibroblasts were selected because they are readily cultivated and radio-labeled and are available from skin explants of a variety of species. Autoradiograms of radiolabeled cellular proteins are more diverse and easily analyzable for molecular systematics than alternatives such as silver-stained serum or erythrocyte protein patterns. We have achieved nearly 100% success rates at minimal discomfort and risk to human subjects by establishing cultures from 3 mm punch biopsies from the upper buttock. Local lidocaine anesthesia is used. Samples are collected following informed consent and under an approved human research protocol. Other species are sampled by dart gun8 or while sedated. Skin samples can be collected from various body sites without compromising the 2D electrophoresis metric, which does not rely on quantitative differences in protein expression. To comply with the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES), tissues collected from wild and captive exotic animals must be obtained under specific permits issued by the U.S. Fish and Wildlife Service. 

The bioavailability of iron from several organic phosphorus-containing compounds appears to be good. The iron in ferripoly-phosphate protein powder (13) and ferric glycerol phosphate Q, 6) was found to be 92-100% as bioavailable as ferrous sulfate in heme repletion assays with anemic rats and chicks. Morris and Ellis (14) have reported that the iron in monoferric phytate was utilized by rats as well as the iron in ferrous ammonium sulfate. While Lipschitz, et al. (15) have reported that dogs absorbed radio-labelled iron from a small dose (1.5 mg iron) of monoferric phytate one-half as well as they absorbed iron from ferrous sulfate. 

---