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Measurement of protein solutions concentrations by biuret protein assay

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Introduction

´╗┐Title: Measurement of protein solutions concentrations by biuret protein assay Objective: To study the principle a protein assay. To determine the absorbance rate of protein at different concentration by using spectrophotometer. To determine the concentration of unknown sample from the graph. Introduction: Protein quantitation is often necessary before processing protein samples for isolation, separation and analysis by chromatographic, electrophoresis and immunochemical techniques. Depending on the accuracy required and the amount and purity of the protein available, different methods are appropriate for determining protein concentration. Instead, several colorimetric, reagent-based protein assay techniques have been developed that are used by nearly every laboratory involved in protein research. Protein is added to the reagent, producing a colour change in proportion to the amount added. Proteins concentration is determined by reference to a standard curve consisting of known concentrations of a purified reference protein. Most pure protein solutions containing 1 mg/mL of protein have an absorbance of about 1.0 when the light path is 1 cm. This method is simple, rapid, and allows for full recovery of the protein. However, many other biochemicals absorb near this wavelength, making an accurate quantitation difficult. Furthermore, different proteins absorb to different extents depending upon their aromatic amino acid content. To avoid these problems, the Biuret test was developed. ...read more.

Middle

The value can be assumed approximately to 5 mg/mol. ________________ ________________ Discussion: The biuret reagent was used for both qualitative and quantitative analysis of protein. The biuret method depends on the presence of peptides bonds in proteins. The solution of proteins is treated with cupric ions (Cu2+) in a moderately alkaline medium, a purple coloured Cu2+ - peptide complex is formed that was measured quantitatively by spectrophotometer. So, biuret reagent is alkaline copper sulphate solution. The intensity of the colour produced is proportional to the number of peptide bonds that are reacting, and therefore to the number of protein molecules present in the reaction system. The reaction don't occur with amino acids because the absence of peptide bonds, and also that with di-peptide because presence of only one peptide bond, but do with tri-, oligo-, and poly-peptides. Biuret reaction needs presence of at least two peptide bonds in a molecule. The reaction occurs with any compound containing at least two bonds of: -HN-CO- , -HN-CH2- , -HN-CS- . Because proteins differ in their amino acid compositions, each one responds somewhat differently in each type of protein assay. Therefore, the best choice for a reference standard is a purified, known concentration of the most abundant protein in the samples. ...read more.

Conclusion

Another interfering factor would be ratio of amino acids presents in different proteins which absorb at different rate as well. Next is Lowry assay method, is based on the reduction by protein of the phosphomolybdic-tungstic mixed acid chromogen in the Folin-Ciocalteu?s phenol reagent, resulting in an absorbance maximum at 750 nm. The Folin-Ciocalteu?s phenol reagent reacts primarily with tyrosine residues in the protein, which can lead to variation in the response of the assay to different proteins. The method is sensitive to interfering substances; a procedure for precipitation of the protein from the test specimen need be to used. Most interfering substances cause a lower colour yield. However, some detergents cause a slight increase in colour. A high salt concentration can cause precipitation to form. The Bradford assay, is based on the absorption shift from 470 nm to 595 nm observed when the brilliant blue G-250 dye binds to protein. The Coomassie Brilliant Blue G-250 dye binds most readily to arginyl and lysyl residues in the protein, which can lead to variation in the response of the assay to different proteins. Slow precipitation of the dye will occur during storage of the reagent. Therefore, the reagent must be filtered before use. The relationship of absorbance to protein concentration is nonlinear. However, if the standard curve concentration range is sufficiently small, it will approach linearity. Conclusion: Biuret protein assay is a suitable method for measurement of protein solutions concentration. ...read more.

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A thorough consideration of the use of the biuret test for protein assays. Good use of A level scientific language throughout.

Marked by teacher Adam Roberts 29/05/2013

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