Although the mechanism of color formation for the lowry assay is similar to that of the bca protein assay, there are. 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. Based off the chemistry 221 laboratory manual, the reaction is concentration dependent and. This protocol describes a method of quantifying protein that is a variation of the lowry assay. Bicinchoninic acid bca assay in low volume sciencedirect. It is important to note that the estimated protein levels using this reagent can sometimes be skewed in the presence of phospholipids. 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. Bca bicinchoninic acid protein assay is watersoluble and strongly absorbs light at 562nm in a linear fashion with increasing protein concentration. In test tubes or 96well plates, the relationship between protein concentration and absorbance is nearly linear over a wide working. Interference by lipids in the determination of protein. Instructions bca protein assay reagent kit pdf book. Some of the variables and the interferences which affect the reaction kinetics of proteins with bicinchoninic acid bca 4,4dicarboxy2,2biquinoline have been studied. Protein assay technical handbook thermo fisher scientific.
The bicinchoninic acid bca protein assay is suitable for quantifying protein solutions in 1ml assays or in microwells. Insitu microvolume bicinchoninic acid protein assay. Bca protein assay kit ab102536 provides a simple, rapid, detergent tolerant up to 5% procedure for determining the concentration of proteins in solution. The bca assay was demonstrated using two microvolume formats. Pdf measurement of protein using bicinchoninic acid m. Bicinchoninic acid assay an overview sciencedirect topics. Rapid solidphase determination of cereal protein using bicinchoninic acid kinyu chan and bruce p. The absorbance is directly proportional to the amount of protein present in the solution and it. Nacetylaspartate availability is essential for juvenile. The most common protein assay reagents involve either proteindye binding chemistry coomassiebradford or proteincopper chelation chemistry biuretbca. More sensitive than lowry and less subject to interferences. The color produced is stable and increases in a linear fashion over a broad working range of increasing protein concentration. First is the biuret reaction, whose faint blue color results from the reduction of cupric ion to cuprous ion. The bca is less prone to interference than the lowry procedure and is more sensitive.
Those peptide groups are then free to bind another molecule of cupric ion. Bicinchoninic acid kit for protein determination bca. A new protein assay may be adequate in different experiments beyond or instead of the wellknown standard protocols e. 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. 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. Studies of electrode reactions and coordination geometries. Instead, bicinchoninic acid based assay was, indeed, distinctly affected by the presence of nanoparticleiron in suspension 0.
The bicinchoninic acid assay bca assay, also known as the smith assay, after its inventor. The response of the bca chromophore to various interfering substances has provided insight into the mechanism of protein quantitation by. The first step is the chelation of copper with protein in an alkaline environment to form a light blue complex. Qubit, bradford or bicinchoninic acid in related conceptions. Pdf bicinchoninic acid bca assay in low volume researchgate. Pdf the bca assay is a colorimetric method for estimating protein. Protocol for bca protein assay using a nanodrop 20002000c spectrophotometer subject. Bicinchoninic acid protein assay kit bca1 technical. 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. Receive a free secondary antibody with your primary antibody purchase. Colorimetric protein assays thermo fisher scientific. Chemistry of protein assays thermo fisher scientific us. Us4839295a measurement of protein using bicinchoninic. The bicinchoninic acid bca assay was invented in 1985 by paul k.
The lowry protein assay is a biochemical assay for determining the total level of protein in a solution. This protocol describes a method of quantifying protein that is a variation of the. The two assays are of similar sensitivity, but since bca is stable under alkali conditions, this assay has the advantage that it can be. This procedure is very applicable to microtiter plate methods. The bicinchoninic acid bca assay, first described by smith et al. Bicinchoninic acid kit and bovine serum albumin bsa were obtained from sigmaaldrich. Otherwise, contact supplier for specific applications. Bicinchoninic acid bca protein assay photometric method reading at 562 nm. The bicinchoninic acid bca protein assay primarily relies on two reactions. Quantifying protein by bicinchoninic acid csh protocols. The bca is used for the same reasons the lowry is used. The bicinchoninic acid bca assay for protein quantitation.
With the exception of tyrosine and tryptophan, free amino acids will not produce. Since bca is stable, it is incorporated in the reagent formulation at. Standard curve of net absorbance versus protein sample concentration incubate at 37. Bicinchoninic acid bca protein assay, gbiosciences vwr. The bicinchoninic acid assay bca, also referred to as the smiths assay, is a biochemical assay designed by paul smith in 1985. The bicinchoninic acid bca protein assay is a highly sensitive colorimetric assay that is. Ensure that the wells are free of air bubbles and that the bottom of the plate is clean.
Tools and reagents for improved quantitation of total or. Lipidstainingwasperformed in fixed cells 10% formalin in pbs, 30 min using oil red o 0. Box 117 industriezone iii rockford, il 61105 industrielaan 27 usa 9320 erembodegemaalst 815. Compared to native protein absorbance at 280 nm, bca has improved sensitivity and good specificity for protein with minimal interference from. According to bradford, it is similar to the lowry protein assay, bradford protein assay or biuret reagent. 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 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. 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. The bicinchoninic acid bca protein assay is a highly sensitive colorimetric assay that is compatible with detergent solubilized protein solutions. The bicinchoninic acid bca 2 assay is a colorimetric method that is commonly employed to estimate the concentration of protein in a sample. Bicinchoninic acid kit protein determination is one of the most common operations performed in biochemical research. 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. Test for protein quantification linkedin slideshare. Please login or register for free to view full text.
The bicinchoninic acid assay bca assay, also known as the smith assay, after its inventor, paul k. Synonyms bicinchoninic acid protein assay reagent a intended use refer to the instruction booklet for proper and intended use. Rapid solidphase determination of cereal protein using. The first format was the minibca assay recommended by the nanodrop technical manual 2.
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