The bicinchoninic acid bca 2 assay is a colorimetric method that is commonly employed to estimate the concentration of protein in a sample. The two assays are of similar sensitivity, but since bca is stable under alkali conditions, this assay has the advantage that it can be. A new protein assay may be adequate in different experiments beyond or instead of the wellknown standard protocols e. Some of the variables and the interferences which affect the reaction kinetics of proteins with bicinchoninic acid bca 4,4dicarboxy2,2biquinoline have been studied. The bicinchoninic acid bca assay for protein quantitation. First is the biuret reaction, whose faint blue color results from the reduction of cupric ion to cuprous ion. Lipidstainingwasperformed in fixed cells 10% formalin in pbs, 30 min using oil red o 0. The most common protein assay reagents involve either proteindye binding chemistry coomassiebradford or proteincopper chelation chemistry biuretbca. Standard curve of net absorbance versus protein sample concentration incubate at 37. The bca assay was demonstrated using two microvolume formats. Bicinchoninic acid protein assay kit bca1 technical. Protein assay technical handbook thermo fisher scientific. Bca bicinchoninic acid protein assay is watersoluble and strongly absorbs light at 562nm in a linear fashion with increasing protein concentration. Bicinchoninic acid kit for protein determination bca.
Compared to native protein absorbance at 280 nm, bca has improved sensitivity and good specificity for protein with minimal interference from. The first step is the chelation of copper with protein in an alkaline environment to form a light blue complex. Bicinchoninic acid bca protein assay, gbiosciences vwr. Box 117 industriezone iii rockford, il 61105 industrielaan 27 usa 9320 erembodegemaalst 815. Rapid solidphase determination of cereal protein using. Us4839295a measurement of protein using bicinchoninic. The bicinchoninic acid bca protein assay is a highly sensitive colorimetric assay that is compatible with detergent solubilized protein solutions. Insitu microvolume bicinchoninic acid protein assay february 26. Bicinchoninic acid bca protein assay photometric method reading at 562 nm. Synonyms bicinchoninic acid protein assay reagent a intended use refer to the instruction booklet for proper and intended use. Pdf bicinchoninic acid assay protein quantification. The standard bca assay uses large volumes of both reagents and samples.
Bca protein assay kit ab102536 provides a simple, rapid, detergent tolerant up to 5% procedure for determining the concentration of proteins in solution. Based off the chemistry 221 laboratory manual, the reaction is concentration dependent and. The bicinchoninic acid bca assay was invented in 1985 by paul k. Stoscheck 1990 has suggested that the bca assay will replace the lowry because it requires a single step, and the color reagent is. The color produced is stable and increases in a linear fashion over a broad working range of increasing protein concentration. Chemistry of protein assays thermo fisher scientific za. The bicinchoninic acid bca protein assay primarily relies on two reactions. Bca protein assay kit is used for the determination of total protein. Quantifying protein by bicinchoninic acid csh protocols. The bicinchoninic acid assay bca, also referred to as the smiths assay, is a biochemical assay designed by paul smith in 1985. Qubit, bradford or bicinchoninic acid in related conceptions.
This procedure is very applicable to microtiter plate methods. Receive a free secondary antibody with your primary antibody purchase. Smith at the pierce chemical company, now part of thermo fisher scientific, is a biochemical assay for determining the total concentration of protein in a solution 0. Reagents bicinchoninic acid solution, catalog number b9643 reagent a is a 1,000 ml solution containing bicinchoninic acid, sodium carbonate, sodium tartrate, and sodium bicarbonate in. Interferences attributed to glucose, ascorbic acid, and uric acid in this reaction were significantly reduced by performing a twopoint assay and by including borate ions in the buffer. Storage and stability 12 months at room temperature introduction quantumprotein bicinchoninic acid protein assay1,2,3 kit is formulated for the colorimetric detection and quantitation of total protein. The bicinchoninic acid bca protein assay is a highly sensitive colorimetric assay that is compatible with detergent solubilized protein solutionsaccurately quantifying total protein concentration is a key to most experiments and is often necessary for isolation, separation, and analysis of proteins by biochemical methods. With the exception of tyrosine and tryptophan, free amino acids will not produce. Otherwise, contact supplier for specific applications. This protocol describes a method of quantifying protein that is a variation of the. The bca is less prone to interference than the lowry procedure and is more sensitive. Chemistry of protein assays thermo fisher scientific us. According to bradford, it is similar to the lowry protein assay, bradford protein assay or biuret reagent. Although the mechanism of color formation for the lowry assay is similar to that of the bca protein assay, there are.
Protocol for bca protein assay using a nanodrop 20002000c spectrophotometer subject. Interference by lipids in the determination of protein. Rapid solidphase determination of cereal protein using bicinchoninic acid kinyu chan and bruce p. Ensure that the wells are free of air bubbles and that the bottom of the plate is clean. The first format was the minibca assay recommended by the nanodrop technical manual 2. Introduction the modulus by turner biosystems and the bca protein assay kit from pierce provides a simple method for quantifying protein over a large range. The protein protocols handbook, second edition aims to provide a crosssection of analytical techniques commonly used for proteins and peptides, thus providing a benchtop manual and guide for those who are new to the protein chemistry laboratory and for those more established workers who wish to use a technique for the first time. The response of the bca chromophore to various interfering substances has provided insight into the mechanism of protein quantitation by. Instead, bicinchoninic acid based assay was, indeed, distinctly affected by the presence of nanoparticleiron in suspension 0.
The total protein concentration is exhibited by a color change of the sample solution in proportion to protein concentration, which can then be measured using colorimetric techniques. The bicinchoninic acid assay bca assay, also known as the smith assay, after its inventor, paul k. Bicinchoninic acid kit protein determination is one of the most common operations performed in biochemical research. The bicinchoninic acid bca assay is available in kit form from pierce rockford, ill. Please login or register for free to view full text. Test for protein quantification linkedin slideshare. The micro bicinchoninic acid bca protein assay is a highly sensitive colorimetric assay that is compatible with detergent solubilized protein solutions and is a modification of the bicinchoninic acid bca protein assay for dilute protein samples 0. Pdf measurement of protein using bicinchoninic acid m. Tools and reagents for improved quantitation of total or. The bicinchoninic acid bca protein assay is suitable for quantifying protein solutions in 1ml assays or in microwells. The lowry protein assay is a biochemical assay for determining the total level of protein in a solution. The bicinchoninic acid assay, also known as the bca assay and smith assay after its inventor paul smith, is a biochemical assay for determining the total concentration of protein in a solution. Those peptide groups are then free to bind another molecule of cupric ion.
The bicinchoninic acid bca assay, first described by smith et al. Pdf bicinchoninic acid bca assay in low volume researchgate. The bicinchoninic acid bca protein assay is a highly sensitive colorimetric assay that is. Since bca is stable, it is incorporated in the reagent formulation at. The absorbance is directly proportional to the amount of protein present in the solution and it. This protocol describes a method of quantifying protein that is a variation of the lowry assay.
Bicinchoninic acid bca assay in low volume sciencedirect. The bca is used for the same reasons the lowry is used. Nacetylaspartate availability is essential for juvenile. Insitu microvolume bicinchoninic acid protein assay. The bicinchoninic acid assay bca assay, also known as the smith assay, after its inventor. When it is necessary to determine the total protein concentration in a. Bicinchoninic acid assay an overview sciencedirect topics. More sensitive than lowry and less subject to interferences. In test tubes or 96well plates, the relationship between protein concentration and absorbance is nearly linear over a wide working.
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