The bicinchoninic acid assay bca assay, also known as the smith assay, after its inventor, paul k. To evaluate the performance of each of the purification methods performed earlier, purified omvs were evaluated by several methods as summarized in table 1. The bicinchoninic acid bca assay is available in kit form from pierce rockford, ill. A typical bca assay standard curve with samples ranging from 0. A purple colored reaction product is formed by the chelation of two molecules of bca with one cuprous ion. The data sheet of the bca assay says that up to 5% of tween80 do not interfere with the assay, but we have problems even with 0. A 25l sample volume is recommended for the microplate procedure of the bca protein assay. The bicinchoninic acid bca protein assay is based on a twostep reaction in one tube. Microplate bca assay for wholecell lysate entered by. Bca total protein assay kit gene and cell technologies. The thermo scientific pierce bca protein assay is a detergentcompatible formulation based on bicinchoninic acid bca for the colorimetric detection and quantitation of total protein. This protocol makes use of thermo scientifics bca protein assay in a 96well tissue culture plate format to measure the amount of protein in small volumes of sample.
This was done by analyzing the precision, accuracy, linearity and range of the method. An adaptaon of the bca protein assay kit, the micro bca kit has been opmized for use with dilute protein samples 0. The pierce bca assay can be used to assess yields in whole cell lysates and affinitycolumn fractions, as well as to monitor protein contamination in industrial applications. Pierce bca protein assay kit, sufficient reagents for 500 testtube or 5000 microplate assays. The micro bca protein assay kit uses concentrated reagents and a protocol that utilizes an extended incubation time at an elevated temperature 60c, test tube procedure only.
The size distribution and concentration of purified omvs were. Download the pdf or microsoft word versions of this protocol. Introduction beers law tells us that the light absorbance a of a solution is proportional to the. Total protein content was measured by a standard bicinchoninic acid assay bca assay. The bca is used for the same reasons the lowry is used. Compatibility chart for bca kit the amount listed is the maximum amount of material allowed in the protein sample without causing a noticeable interference, when 20 ul protein sample is used for bca assay as described in the standard assay. Incompatible substances amount compatible buffer systems nacetylglucosamine 10 mm in pbs, ph 7. Bca assay can be performed in a microtiter plate format or test tube format. Standard protein quantification methods, such as bicinchoninic acid assay bca will suffice. Biomedical and biological sciences 59,406 views 24.
The takara bca protein assay kit is a highly sensitive colorimetric assay that is compatible with detergent solubilized protein solutions. How to do bca bradford protein quantification assay for. Compared to other methods, the bca assay is one of the most sensitive it can detect proteins at concentrations as low as 5 ugml. Bicinchoninic acid protein assay kit bca1 technical. Compatibility of pierce bca protein assay with promega lysis. The bichinchoninic acid bca assay is used to estimate the total protein concentration in a sample. When ready to run the assay, equal amounts of protein should be loaded per condition. The thermo scientific pierce bca protein assay kit is a detergentcompatible formulation based on bicinchoninic acid bca for the colorimetric detection and quantitation of total protein. The two most commonly used methods for protein quantification are the bca method, bradford coomassie brilliant blue method. Piercea protein assay kit bc thermo fisher scientific. Bca protein assay kit, sufficient reagents for 500 test tube or 5,000 microplate assays.
Studies of the bca assay for determination of total protein in allergen extracts title swedish abstract the aim of this thesis has been to evaluate and to optimize the bca protein assay for quantification of total protein. This reaction produces a blue color with an absorbance peak at 562 nm. The bicinchoninic acid bca assay, first described by smith et al. Both a standard assay for concentrated proteins and a micro assay for dilute protein solutions are described below. Jun 19, 2018 the principle of lowry assay, biuret assay, and bicinconinich bca assay protein assays duration. The bicinchoninic acid bca assay for protein quantitation. The bicinchoninic acid bca protein assay is a highly sensitive colorimetric assay that is compatible with detergent solubilized protein solutions. The bca working reagent was prepared just prior to use as per the manufacturers recommendations.
The two assays are of similar sensitivity, but since bca is stable under alkali conditions, this assay has the advantage that it can be. Bicinchoninic acid assay an overview sciencedirect topics. Combine samples and lowry assay reagents in polystyrene tubes 2. This colorimetric assay provides the high sensitivity and linearity associated with the bca assay, but in a fraction of the time it takes to perform a standard bca assay. Objective utilizing beers law, known concentrations of bovine serum albumin bsa are reacted with bicinchonic acid bca to create a standard curve of absorbance vs. The two assays are of similar sensitivity, but since bca is stable under alkali conditions, this. This protocol makes use of thermo scientifics bca protein assay in a 96well. The remaining kit components should be stored at room temperature. Add 25 l of each bsa standard and protein samples into microtiter plate wells. The pierce bca protein assay is a detergentcompatible formulation based on bicinchoninic acid bca for the colorimetric detection and quantitation of total protein. These and others changes like the bca working reagent to protein sample ratio affect the net absorbance values. It uses the same copperchelating technology as the wellknown traditional pierce bca protein assay and provides comparable accuracy bu. Microplate bca assay for wholecell lysate entered by kevin janes janes lab protocols 31209 1 1.
The protocols described here are for guidance only. Stoscheck 1990 has suggested that the bca assay will replace the lowry because it requires a single step, and the color reagent is stable under alkaline conditions. 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. In comparison to the classical methods by bradford and lowry, the bca assay suffers less variability due to protein sequence, and is less prone to interference by nonprotein substances.
Add 10l of unknown sample to each of the appropriate wells. Protein assay standard curves and data processing fundamentals are discussed in detail. The bca protein assay kit can be used to measure the protein concentration of lysates or homog enates, in microplate format. Studies of the bca assay for determination of total.
It uses the same copperchelating technology as the wellknown traditional pierce bca protein assay and provides comparable accuracy but with a 5min, room temperature incubation. Bca is sensitive and has a broad dynamic range capable of measuring protein concentrations of 0. Standard protocols for bicinchoninic acid bca assays using vials or. Pierce bca protein assay kit thermo fisher scientific. I had similar problems with the microplate so i switched to what they call the test tube procedure, or standard protocol. The reaction reaches 90% of its maximum color development within 15 minutes thereby. It is recommended that the bca compatibility reagent be used to decrease interference from reducing agents, chelators, detergents, and other common ingredients found in most lysis buffers. Herein, we used the pierce bca protein assay kit thermo scientific, catalog no. Watched so many videos but dont understand the concept or steps. Competitive binding to cuprous ions of protein and bca in the. Compatibility of pierce bca protein assay with promega. This unit also details highthroughput adaptations of the commonly used protein assays, and also contains a protocol for bca assay of total protein in sdspage sample buffer that is used for equal loading of sdspage gels, which is reliable, inexpensive, and. The thermo scientific pierce bca protein assay kit is a detergentcompatible formulation.
The bici nchoninic acid bca protein assay is suitable for quantifying protein solutions in 1ml assays or in micro wells. A brief description of the principle and the choice of the experiment is as follows bca principle. This is called a minibca assay by the nanodrop manual 2. Insitu microvolume bicinchoninic acid protein assay february 26. The reaction is similar to the welldocumented lowry1 assay, but with the following improvements. Stoscheck 1990 has suggested that the bca assay will replace the lowry because it requires a single step, and the color reagent is. Bicinchoninic acid protein assay on materials used. The pierce rapid gold bca protein assay kit is a fast, twocomponent, highprecision, detergentcompatible assay optimized to determine total protein concentration.
Aug 02, 2010 the principle of lowry assay, biuret assay, and bicinconinich bca assay protein assays duration. The first step is the chelation of copper with protein in an alkaline environment to form a light blue complex. Biomedical and biological sciences 61,514 views 24. Each assay was performed in duplicate, and the results are presented as the average of duplicates. A purple colored reaction product is formed by the chelation of two molecules of. The result is an extremely sensitive colorimetric protein assay in a test tube or microplate assay format. The standard protocol permits either a 30minute incubation at 37c or a 2hour incubation at room temperature. The principle of lowry assay, biuret assay, and bicinconinich bca assay protein assays duration. The bca assay is based on the traditional lowry assay except bicinchoninic acid is used resulting in a very pretty purple color that absorbs at 560 nm. Total dna content was determined by measuring the absorbance at 260 nm. The bca protein assay kit is a detergentcompatible formulation based on bicinchoninic acid bca for the colorimetric detection and quantitation of total protein. The pathlength in each assay is dependent on the assay container cuvettes or multiwell plates andor the reaction volume. This curve is then utilized to determine the concentration of protein in an unknown solution. Compared to most dyebinding methods, the bca assay is affected much less by protein compositional differences, providing greater proteintoprotein uniformity.
The biorad protein assay is a dyebinding assay in which a differential color change of a dye occurs in response to various concentrations of protein. The bicinchoninic acid bca protein assay primarily relies on two reactions. I have found the assay to be fairly sensitive and reliable, however a few points should be noted. Difference between bca and bradford method to determine. Competitive binding to cuprous ions of protein and bca in. Bicinchoninic acid protein assay kit bca1 technical bulletin. Be aware of any hazardous compounds, take precautions where necessary and dispose of any waste in the appropriate manner. Basically i add 50 parts of reagent a to one of b and this is the working reagentwr.
Bca protein assay kit, sufficient reagents for 500 test tube or 5,000. Bca versus biorad protein assay what are the advantages of bca that all the world uses it. Studies of the bca assay for determination of total protein. 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. The thermo scientific pierce bca protein assay kit is a detergent compatible formulation. Add 10l of bsa standards stored in 80c freezer to each of the appropriate wells. The absorbance at 562nm was read in a spectramax 250 plate reader. Please see the attached protocol for additional details. The basis for this assay is the reduction of copper in alkali by protein biuret reaction in the presence of the chromogenic agent bicinchoninic acid. Unfortunately we cannot switch to another protein assay as we. The absorbance is directly proportional to the amount of. The biorad dc protein assay is a colorimetric assay for protein concentration following detergent solubilization. The quantipro bca assay kit is based on the same principals as the bca1 kit, except the quantipro bca kit gives a linear response from 0.
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