Transferrin solution preparation

GHz Fe(III)-transferrin-ll complex at room temperature. The 5 X 10 4M transferrin solution was about 90% saturated with Fe(III) and nitroxyl-labeled malonate (compound II). (B) After the preparation was made 5 X 10 3M in bicarbonate (49). 

In 1993, another immunoassay for the detection of monensin was developed but, unfortunately, was never applied to biological material (91). Quite recently a competitive ELISA and a compatible extraction procedure suitable for screening monensin in poultry liver samples was described (92). In this assay, a polyclonal antiserum raised against a monensin-transferrin conjugate and prepared via an acid anhydride intermediate (93) was used. Significant cross-reactivity with other polyethers commonly used by the broiler industry, such as maduramicin, lasalocid, salinomycin, and narasin, was not found. A detection limit of 3 ppb could be readily attained when liver samples were submitted to extraction with aqueous acetonitrile, partitioning between aqueous sodium hydroxide solution and a hexane-diethyl either mixture, evaporation of the organic phase, and reconstitution in ethanol/sodium acetate solution. 

Obtain a precast SDS-polyacrylamide slab gel or prepare one according to instructions in Experiment 4. The recommended gel is 12°/o acrylamide with a thickness of 0.75 mm. Protein samples are prepared as follows Purified proteins (transferrin, bovine serum albumin, a, -antitrypsin, a-lactalbumin from Experiment 4, and molecular weight standards) are supplied in Tris buffer, pH 6.8 solutions at a concentration of 1 mg/mL. Sera samples have been diluted and are ready for use. Prepare protein samples for electrophoresis in 0.5-mL microcentrifuge tubes with attached caps. Label the tubes from 1 to 5 as below or per your Instructor s directions. 

Cysteine residues in transferrin were reduced and alkylated in a similar manner as that done for solution samples p). Modiflcation for samples prepared with ProSorb cartridge can be performed in the same ProSorb cartridge before membrane removal, while modifications were performed in an Eppendorf tube for the electroblotted samples. The membranes were incubated 15 minutes at room temperature in a 0.25 M Tris/HQ and 6 M Guanidine hydrochloride buffer containing 1 ml of mercaptoethanol and followed by the addition of 1 ml of 4-vinyl pyridine for another 15 minutes. The membnmes were washed thoroughly with 0.1% TFA afterwards. 

As an important example of a biochemically relevant species, Se-labeled selenomethionine can be prepared, for example, by isolation from selenized yeast, grown in a Se-labeled selenite solution. After digestion of the yeast with pro-tease/lipase solution, Se-labeled selenomethionine is isolated by size-exclusion chromatography (SEC) [72, 73]. Another important example is the synthesis of Fe-labeled transferrin [74]. The synthesis of other biologically relevant molecules, especially those of higher molecular weight, is usually too complicated, and the species-unspecific spiking mode is more convenient in these cases. 

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