The biuret test is a chemical assay that detects the presence of proteins in a sample. The test relies on a color change to confirm the presence of proteins. Biuret is a chemical compound with the chemical formula h 2 nco 2 nh. Lowry assay, biuret assay, bradford assay, and bca assay. Biuret reagent was used to measure the quantity of protein in all samples. Biuret protein assay introduction substances containing more than two peptide bonds form a purple complex with copper salts in alkaline solution. Links to pubmed are also available for selected references. Revised fall 2010 biuret protein assay augusta university.
One commonly used method for determining the total protein in a sample is the biuret method. D, thermo fisher scientific, madison, wi, usa subject. In the presence of peptides, a copperii ion forms mauvecolored coordination complexes in an alkaline solution. The copper ions bind to amide groups of proteins and produce a blue pigment that can be quantified using a spectrophotometer griner 2003. Get a printable copy pdf file of the complete article 1. Transfer to suitable cuvettes and record the absorbance at 540 nm for test, standards, and blank. A rapid procedure for the estimation of total protein, true albumin, total. Seven samples of corn and corn coproduct were randomly collected from starch and ethanol plants. First documented in 1951, lowry assay was an oftencited. Determination of total protein content in gelatin solutions with the lowry or biuret assay article in journal of food science 718. Information from its description page there is shown below.
The term biuret also describes a family of organic compounds with the functional grouphnco 2 n. For the test solutions of 1% albumin, it turns blue color to purple. Transfer to suitable cuvettes and record the absorbance at 540 nm for test. Recovery is complete and absorbancy linear with both concen tration of the soluble. This assay therefore requires more protein than other common methods such as the bca, lowry and bradford assays.
For the test sample, 1ml of the protein sample was added to 4. Cuprous ions are produced by the reduction of cupric ions by proteins in alkaline solutions. As the number of peptide increases anon, 2014, more complexes are formed thus causing the colour to deepen. In the presence of peptides, a copperii ion forms mauvecolored coordination. Pdf the concentration of dissolved protein was determined separately with biuret, bicinchoninic acid and bradford. The key component in this assay is bicinchoninic acid bca which reacts with cuprous ions to generate an intense purple color at 562 nm. Recovery is complete and absorbancy linear with both concen tration of the soluble crystalline serum albumin standard and the volume of homogenate of a variety of tissues. The copper ions bind to amide groups of proteins and produce a blue pigment. The biuret assay was commonly employed well into the 1980s and is still in use because it is so convenient and inexpensive to prepare and easy to use. Its a funny thing, but the biuret test doesnt involve the chemical biuret, which is derived from urea. Prepare a reagent blank by adding 100 l of water to a sixth test tube different test tube filled with biuret reagent. The working range for the biuret assay is 5160 mgml, which is adequate for some types of industrial applications but not nearly sensitive enough for most protein research needs.
The biuret reaction can be used to assay the concentration of proteins because peptide bonds occur with the same frequency per amino acid in the peptide. We report an evaluation of the first rate biuret assay of protein in human. The protein sample, irrespective of its composition, is measured through absorbance spectroscopy at 540 nm in conjunction with a known protein concentration sample. Determination of the sensitivity range of biuret test for. The working range for the biuret assay is 5160mgml the presence of 1 or 2 amino acid is. In the biuret protein assay procedure, copper sulfate dissolved in alkaline solution is added to a protein mixture. The biuret reagent 1 is prepared by adding sodium hydroxide to a copper sulphatesodium potassium tartrate solution. One of the simplest and most common is the biuret protein assay. The biuret test, also known as piotrowskis test, is a chemical test used for detecting the presence of peptide bonds. The protein concentration affects the intensity of the color, and the color will be more intense with more proteins. There are a number of very good and very sensitive protein assay methods. In addition to the standard curve, you will be assaying one of seven different protein samples of unknown concentration. The working range for the biuret assay is 5160mgml the presence of 1 or 2 amino acid is insufficient for the biurets test to work because it requires at least 2 peptide bonds for the biurets test to work.
There are very few interfering agents ammonium salts being one. A practical method for extending the biuret assay to protein. The biuret assay is thereby chosen and further developed in the present study to provide an alternative rapid protein determination method for corn or cornbased products. To perform a biuret test, you may need to homogenize or blend the sample to achieve a uniform consistency. Determination of biuret and urea by highperformance liquid. For the biuret test, the test solutions of soya bean turns blue color to light purple because the presence of peptide bond. Biuret protein assay the principle of the biuret assay is similar to that of the lowry. The intensity of the color, and hence the absorption at 540 nm, is directly proportional to the protein concentration, according to the beerlambert law. Rarely, if ever, will the test sample produce an assay response that corresponds exactly to one of the specific standard samples. Biuret was subjected to highperformance liquid chromatography on a reversedphase column 250. If the solution turns purple it means that protein is present.
Assay principle in alkaline medium the copper reacts with the peptide bonds of proteins to form the characteristic pink to purple biuret complex. In alkaline medium the copper reacts with the peptide bonds of proteins to form the characteristic pink to purple biuret complex. The protein assay is based on the interaction of cupric ions with protein in an alkaline solution and is commonly referred to as the biuret assay. Advantages of the kdsbca assay over the bradford assay for protein quantification in white wine and grape juice am. There are very few interfering agents ammonium salts being one, and layne 1957 reported fewer deviations than with the lowry or ultraviolet absorption methods. The method described here uses quantitative biuret solution which will give a good estimate of protein concentrations in the range 0.
Determination of total protein content in gelatin solutions. The absorbance was measured at a wavelength of 750nm for lowry, 540 nm for biuret and 595 nm for. However, it involves a single incubation of 20 minutes. This lab activity is designed to teach students the principl es behind a common protein estimation assay known as the biuret protein assay. Spectrophotometric determination of total protein biuret.
The bca protein assay has been advertised as an alternative to the lowry assay. For the control, 1ml of distilled water was added to 4. C474 c479 october 2006 with 1,500 reads how we measure reads. Background the determination of protein concentration is an essential technique in all aspects of protein studies and proteomics. There are very few interfering agents ammonium salts being one such agent, and layne. The biuret assay, also known as the piotrowski test, is a biochemical assay that allows one to accurately quantify protein concentration within the range of 5150 mgml. A rapid biuret procedure is described which avoids the turbidity that occurs with protein analysis of intact fatty tissues. There are very few interfering agents ammonium salts being one such agent, and layne 1957 reported fewer deviations than with the lowry or ultraviolet absorption methods. The bca protein assay combines the proteininduced biuret reaction see. Spectrum diagnostics total protein reagent is intended for the in. The biuret test detects peptide bonds, and when they are present in an alkaline solution, the coordination complexes associated with a copper ion are violet in color. For last method, bradford, is used as a reagent coomassie brilliant blue g250. The biuret reaction was applied to a quantitative colorimetric test for milk serum. Biuret test the twelve protein standard solutions were subjected to biuret test.
Therefore, a method is needed to calculate or interpolate between the standard sample points. The biuret protein assay was published as a method to determine protein concentration in the 1940s, although the reaction itself was studied as long ago as the early 19th century. Biuret reagent contains copper ions in a basic solution. The term biuret also describes a family of organic compounds with the. Rarely, if ever, will the test sample produce an assay response that corresponds exactly to one of the specific standard. Biuret test or biuret reagent human biochemistry, mcqs. In this experiment, we had used biuret assay and lowry assay to determine.
Biuret protein assay biochemistry biotechnology methods. The conh bond peptide in polypeptide chain reacts with copper sulphate in an alkaline medium to give a purple colour which can be measured at 540 nm. Learn vocabulary, terms, and more with flashcards, games, and other study tools. Full text full text is available as a scanned copy of the original print version. In the biuret protein assay procedure, copper sulfate dissolved in alkaline solution is added to a protein. The biuret and lowry1 procedures are methods for protein determination. The amount of color produced is proportional to protein. Proteins which contain larger mole fractions of tyrosine and tryptophan will give higher absorbance values than those devoid of or containing. The latter, though more sensitive, is used for investigative work and is limited by poor stability of combined reagents, nonreproducibility of color, especially at low protein concentration, and non. Commons is a freely licensed media file repository. This reaction is dependent in part on peptide bonds and not solely on amino acid moieties. The latter, though more sensitive, is used for investigative work and is limited by poor. A qualitative test can be performed simply by adding equal quantities of 1% sodium or potassium hydroxide and a 1% solution of copper sulphate to the sample. The biuret method for the determination of total protein using an evolution array 8position cell changer author.
The name of this assay is somewhat confusing because assaying for proteins using this. There are however, several major disadvantages to this method. Thus, the biuret reaction is the basis for a simple and rapid colorimetric reagent of the same name for quantitatively determining total protein concentration. Total protein biuret reagent ec rep spectrum diagnostics. The principle of the biuret assay is similar to that of the lowry, however it involves a single incubation of 20 min.
The estimation of protein by the biuret and greenberg methods. Four major protein assay protocols have been extensively used in the literature. The method described here uses quantitative biuret solution which will give a good. Although the mechanism of color formation for the lowry assay is similar to that of. This is an experience shared by other investigators 10,30,35 but it should be noted that this opinion is not universal 3638. Principle of the biuret test chemistry stack exchange. Pdf digital photometric determination of protein using biuret. The key component in this assay is bicinchoninic acid bca which reacts with cuprous ions to. Sodium potassium tartarate prevents copper hydroxide precipitation, and potassium iodide prevents the autoreduction of copper. Spectrophotometric determination of total protein biuret method. This can be used to quantify proteins in the concentration range from 0.