Bovine serum albumin is a model protein which has been conventionally used as protein standard and in many areas of biochemistry, pharmacology, medicine, etc. Radioiodination procedure for bovine serum albumin employing chloramine-T as an oxidant with slight modification was evaluated critically to establish the optimal conditions for the preparation of radiolabeled tracer (125I-BSA) with required specific activity without impairing the immunoreactivity and biological activity. Optimized radioiodination procedure involving 10 µg of chloramine-T along with 20 µg of sodium metabisulphite with 60 seconds incubation at 2oC yielded 125I-BSA with high integrity.
Key words: Bovine serum albumin, chloramine-T, radiolabeled, tracer
Serum albumin is the most abundant globular protein in the plasma and synthesized by the liver using dietary proteins. The physiochemical properties and structure of serum albumin is well studied and hence it serves as a model protein for several researchers [1-3]. Radioiodinated bovine serum albumin (BSA) is used in many areas of biochemistry, pharmacology and medicine.125I-BSA has been used conventionally in the tannin precipitation studies[4]. Hence, a well standardised procedure for the preparation of radioiodinated BSA could be useful for a variety of studies involving in-vitro quantification.
Although several methods for labeling 125I are available, radioiodination employing chloramine-T method is the most common method to introduce a radioactive atom into a large biological molecule [5]. The reaction starts with radioactive iodide anion, which is then oxidized using chloramine-T to a more reactive oxidation state. This reacts principally with aromatic groups in protein mainly phenolic tyrosine. The reac...
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...ubunit structure of the protein [8]. The damage caused due to radioiodination was experimentally proved by isoelectric focusing studies [9]. When conditions for reagent concentration was optimized, ideally for every one molecule of oxidant two molecules of reductant is needed to stop the reaction. The result was consistent with the previous radioiodination techniques studied for different protein molecules. It was demonstrated that when incubation time was increased it resulted in damage to the protein. The damage might have caused probably due to increased time of exposure of active iodide species with the protein. Studies on the effect of temperature gave yield of 75% when the reaction was performed at 2oC which is supported by the conclusion that at ambient temperature labeled BSA is in an aggregated form which lowers the iodination yield at low temperature [10].
Abstract/Summary: “Proteins account for more than 50% of the dry weight of most cells, and they are instrumental in almost everything organisms do” (Campbell, 1999). The significance of proteins to the continuation of our biological systems is undeniable, and a study of how to quantify proteins seems an appropriate introduction to our studies of biology. In order to study proteins we must first know how to separate then quantify the amount using basic principles of experimental design such as a standard curve. In this experiment we wish to quantify the amount of previously extracted protein by measuring the absorbance of the unknown amount and determining its concentration by overlaying it against a standard curve of the absorbance of known concentrations of the protein. We used the dye agent Bradford Protein Assay to get an absorbance of 0.078, 0.143, 0.393, 0.473, and 0.527 at the protein’s respective concentrations of 0.28, 0.56, 0.84, 1.12, and 1.40 mg/mL. When a best-fit line was applied to the standard curve, and the absorbance of our unknown concentration (0.317 A) plotted, we estimated a concentration of around 0.84 mg/mL of protein. Our calculations indicated a quantity of 168 mg of protein, which was an approximately 8.96% yield of the projected 1875 mg that was expected. Errors that may have led to this small yield percentage may have stemmed from our previous lab and our initial attempts to extract the desired amount of protein.
J.C. Biro, B. Benyó, C. Sansom, Á. Szlávecz, G. Fördös, T. Micsik, and Z. Benyó; A common periodic table of codons and amino acids. Biochemical and Biophysical Research Communications 306 (2003) 408–415.
Bradford reagent is used to determine the concentration of protein in the experiment, it is a red brown acidic solution in the presence of protein. Bovine serum albumin (BSA) used for various biochemical applications include ELISA which is Enzyme-Linked Immunosorbent Assay, high content of screening assays, western blotting and immunohistochemistry. Furthermore, Bovine Serum Albumin can also be used to determine the quantity of other proteins which is by comparing an unknown quantity of protein to known amount of BSA. In this experiment, Bovine serum albumin (BSA) solution is used as protein concentration standard as the stability of bovine serum albumin will increase signal in the assays and lack of effect in biochemical reactions. Both of these solution are used in part 1 and part 2.
To test for protein, the materials needed are a sample dish, Biuret's solution, and a dropper. Place a small food sample in a dish. Then proceed to add a dropper full of Biuret's solution, if protein is present, solution will turn from a clear to violet or blue. According to the tests, milk, and other milk products showed to have the most protein out of all the
...efore, is to detect and determine phenolic acids in pig blood by examining levels in red blood cells, plasma, albumin, low density lipoprotein (LDL),and very low density lipoprotein (VLDL). It is important to note that when a phenolic compound is present in body fluids, it generally occurs in one or several conjugated forms such as methyl, glucuronide and sulfate, (Proudfoot, Puddey, Beilin, Stocker, & Croft, 1997). There are several methods used to determine bioavailability of phenolic acids and possible metabolites in blood. Some of these methods include electron impact mass spectrometer (EI-MS), atmospheric pressure chemical ionization mass spectrometer (APCI-MS), electro-spray ionization mass spectrometer (ESI-MS), and matrix-assisted laser desorption/ionization mass spectrometer, (S Bertuglia, et al) as well as the more conventional HPLC after identification.
Hankins, Judy BSN,CRNI. The Role of Albumin in Fluid and Electroylte Balance. The Intravenous Nurses Society Volume 29(5), September/October 2006, p250-265
International Union of Biochemistry and Molecular Biology. [Online].; 2002 [cited 2014 April 21. Available from: onlinelibrary.wiley.com/store.
Studying, working, cooking food, caring babies, caring parents, meeting friends…Someone might decide that there is no place for heritage in it and cultural enrichment only has to do with our 2-weeks vacation once a year. But heritage is not static. It is not something available only during travelling. Heritage is all around us: in the receipt of our grandmother’s traditional cookies; in our grandfather’s unusual words, which are disappearing in the modern language; in the way we do gymnastics to avoid health problems in the future…Heritage is not only about gazing, it is about feeling.
In molecular cell biology experiments, accuracy is one of the most important factors in obtaining reproducible results. A luxury not afforded to molecular cell biologists is the ability to easily manipulate samples since the material they deal with is at a microscopic level. Proteins represent a unique opportunity for study because of their intrinsic relation to the central dogma of biology. However proteins are just one subset of macromolecules found in organisms and each protein has its own unique structure that determines its function. When a sample is collected for use in an experiment it is therefore necessary to separate the protein of interest from other macromolecules along with undesired proteins using a series of mechanical and chemical processes. These process of protein purification takes many forms and most rely on the principles of chromatography.
Protein is considered one of the three nutrients that are able to provide calories to the body, with the other two as fat and carbohydrates. Proteins function as building blocks for bones, muscles, cartilage, skin, and blood (US Department of Agriculture, n.d., p. 1). Even after that, they continue to benefit the body in terms of enzymes, hormones, and vitamins. Construction, maintenance, and fixation of body tissues highly de...
EDTA Titrations [homepage on the internet]. No date. [cited 2014 Mar 24]. Available from: http://bionmr.unl.edu/courses/chem221/lectures/chapter-12.ppt.
Plebani, M. (2012). Quality Indicators to Detect Pre-Analytical Errors in Laboratory Testing. The Clinical Biochemist Reviews, 33 (3), 85-88.
One of the first uses of FP was in protein labeling. This allowed the expression and localisation of prot...
The egg white (albumin) is mainly composed of protein molecules. During denaturation process, the protein molecules
Digest the protein to peptides (in gel or solution). Mass spectrometry currently gets limited sequence data from whole proteins, but can easily analyze peptides.