I need to test say four proteins caspase 3,9, pc 51. The bicinchoninic acid bca assay, first described by smith et al. Tools and reagents for improved quantitation of total or. Without protein, the solution is redbrown in its acidic solution. The remaining kit components should be stored at room temperature. Download the pdf or microsoft word versions of this protocol. This protocol makes use of thermo scientifics bca protein assay in a 96well. Unfortunately we cannot switch to another protein assay as we.
Herein, we used the pierce bca protein assay kit thermo scientific, catalog no. The bca protein assay demonstrates higher tolerances towards common interfering. Compared to most dyebinding methods, the bca assay is affected much less by protein compositional differences, providing greater protein to protein uniformity. 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 protocols described here are for guidance only. 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. Be aware of any hazardous compounds, take precautions where necessary and dispose of any waste in the appropriate manner. Thermo scientific pierce micro bca protein assay kit standard protocol. Aug 02, 2010 the principle of lowry assay, biuret assay, and bicinconinich bca assay protein assays duration. The bca bicinchoninic protein assay is a widely used method for colorimetric detection and quantitation of total protein in a solution. Bca assay can be performed in a microtiter plate format or test tube format. The pierce rapid gold bca protein assay kit is a fast, twocomponent, highprecision, detergentcompatible assay optimized to determine total protein concentration. 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. Piercea protein assay kit bc thermo fisher scientific.
Bca protein assay kit for more information before performing the assay. The bicinchoninic acid assay bca assay, also known as the smith assay, after its inventor, paul k. An adaptaon of the bca protein assay kit, the micro bca kit has been opmized for use with dilute protein samples 0. Compare all available bca protein assays features of the bca protein assay kitreducing. Bca protein assay kit ab102536 provides a simple, rapid, detergent tolerant up to 5% procedure for determining the concentration of proteins in solution.
Measuring protein concentration using the bca protein. This was done by analyzing the precision, accuracy, linearity and range of the method. How to do bca bradford protein quantification assay for. Pierce bca protein assay kit reducing agent compatible. The total protein concentration is exhibited by a color change of the sample solution from green to purple in proportion to protein concentration, which can then be measured using colorimetric. How to do bca bradford protein quantification assay for western blotting. Protein assay standard curves and data processing fundamentals are discussed in detail.
Incompatible substances amount compatible buffer systems nacetylglucosamine 10 mm in pbs, ph 7. Bsa bovine serum albumin is used as the standard and samples are. Prepare three to five dilutions of a protein standard, which is representative of the protein solution to be tested. When protein binds, the pka of the dye shifts causing the dye to become blue. The bca assay is the best indirect type of protein assay for protein to protein measurement. Studies of the bca assay for determination of total protein. The bca assay is used to determine the concentration of peptide bonds in a protein sample quantitatively. The bca protein assay kit can be used to measure the protein concentration of lysates or homogenates, in microplate format, prepared with the following buffers. The bca assay is, however, more sensitive to interference from reducing sugars, possibly avidin gelatin e c 0 n n w u z q m o h m q bsa gelatin 200 400 800 1200 protein concentration kgml protein assay using bicinchoninic acid because the protocol allows the sugars more time to reduce cu to cu. It based on bicinchoninic acid bca for the colorimetric detection and quantitation of total protein in the 52,000. The manufacturer of the bca kit advises to use a 562 nm filter, or at most a 590 nm filter. Compatible assay samples that contain up to 5mm dtt, 35mm bme, or 10mm tcep.
Our improved formulation, the bc assay, provides significant benefits over existing colorimetric assays, namely sensitivity, reagent stability, and detergent compatibility. Prepare pierce bovine serum albumin bsa standards 2,000. Dualrange bca protein assay kit is a perfect protein assay method. 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 less variable and less susceptible to detergents than bradford, while bradford assay has a simpler and faster procedure. This bca protein assay kit is a reducing agentcompatible version of our popular pierce bca protein assay. The resulting reaction and color produced is the basis for a common protein quantification method capable of measuring protein concentration over a wide range. Both the incubation time and temperature can be varied over a rather wide range. The thermo scientific pierce bca protein assay kit is a detergent compatible formulation. The bca protein assay kit is a detergentcompatible formulation based on bicinchoninic acid bca for the colorimetric detection and quantitation of total protein. The bca protein assay is a popular method for colorimetric detection and quantitation of total protein. 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. It uses the same copperchelating technology as the wellknown traditional pierce bca protein assay and provides comparable accuracy bu. The bicinchoninic acid bca protein assay is a highly sensitive colorimetric method designed to measure the concentration of a protein of interest compared to a protein standard.
The bca assay has a linear concentration range between 2001,000 g of protein per milliliter. Combine the volumes of reagents a and b specified in the table. Thermo scientific pierce bca protein assay kit reducing agent compatible standard protocol. When ready to run the assay, equal amounts of protein should be loaded per condition. 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. The bichinchoninic acid bca assay is used to estimate the total protein concentration in a sample. Home products molecular biology kits protein assays bca bca bio basic offers high performing kits which maximize quality with significant savings in cost. 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. Bca total protein assay kit gene and cell technologies.
Protocol the following are instructions for the microplate assay format of the pierce bca protein assay kit thermo scientific cat. The bca protein assay kit is a twocomponent, highprecision, detergentcompatible assay reagent set to measure total protein concentration compared to a protein standard. Biomedical and biological sciences 59,406 views 24. This is the protocol to be used in the determination of total protein in brain tissue by the pierce bca method. Pierce bca protein assay kit thermo fisher scientific. Quantipro bca assay kit catalog number qpbca technical bulletin product description protein determination is one of the most common operations performed in biochemical research. Protein solutions are normally assayed in duplicate or triplicate. The bca protein assay is a copperbased protein assay and is also known as the smith assay because it was introduced by paul k.
Tools and reagents for improved quantitation of total or specific proteins. The 1980s saw the introduction by paul smitha of a colorimetric protein assay derived on the biuret reaction using the bicinchoninic acid, known as the bca method. The differences between the bca and bradford protein assay. The kit enables you to measure protein concentration in samples that contain the disulfide reducing agents. The best relative standard to use is one which gives a color yield similar to that of the protein. In any protein assay, the best protein to use as a standard is a purified preparation of the protein being assayed. 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.
If precise quantitation of an unknown protein is required, it is advisable to select a protein. The bicinchoninic acid bca protein assay is a highly sensitive colorimetric assay that. Measuring protein concentration using the bca protein assay. The bici nchoninic acid bca protein assay is suitable for quantifying protein solutions in 1ml assays or in micro wells. 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. This protocol makes use of thermo scientifics bca protein assay in a 96well tissue culture plate format to measure the amount of. Thermo scientific pierce protein assay technical handbook. Bradford protein assay protocol university of san diego. Thermo scientific pierce microplate bca protein assay kit. Acid bca protein assay, also known as the smith assay, is a highly sensitive colorimetric assay that is compatible with detergent solubilized protein solutions. 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 non protein substances.
Bradford dye is easy to use, as well as fast and sensitive, but several compounds can interfere with the assay see the pdf file for the biorad protein assay. Watched so many videos but dont understand the concept or steps. 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. The pierce bca protein assay is a detergentcompatible formulation based on bicinchoninic acid bca for the colorimetric detection and quantitation of total protein. The linear range of this microtiter plate assay is 0. Standard protein quantification methods, such as bicinchoninic acid assay bca will suffice. Review and cite bca assay protocol, troubleshooting and other methodology. The first step is the chelation of copper with protein in an alkaline environment to form a light blue complex. The bicinchoninic acid bca protein assay primarily relies on two reactions.
Bca protein assay kit, sufficient reagents for 500 test tube or 5,000. The result is an extremely sensitive colorimetric protein assay in a test tube or microplate assay format. The bicinchoninic acid bca protein assay is a highly sensitive colorimetric assay that is compatible with detergent solubilized protein solutions. Reagents bicinchoninic acid solution, catalog number b9643 reagent a is a 1,000 ml. Protein assay lab report using the bca assay to determine. 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. Pierce bca protein assay kit, sufficient reagents for 500 testtube or 5000 microplate assays. Bicinchoninic acid assay an overview sciencedirect topics. In the absence of such an absolute reference protein, one must select another protein as a relative standard. Measurement of protein using bicinchoninic acid sciencedirect. Pierce bca protein assays have a unique advantage over the coomassie dyebased assays bradfordthey are compatible with samples that contain up to 5% surfactants detergents, and are affected much less by protein compositional differences, providing greater protein to protein uniformity.
Biovisions bca protein assay kit provides a colorimetric detection and quantification of total protein content even in the presence of detergents. 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. A typical bca assay standard curve with samples ranging from 0. Increasing protein concentrations produce proportionally deeper colors. Measuring the protein concentration of samples is essential for many protein assays, such as western blotting or zymography. Onelab guides you through the workflow and assists in setting up serial dilutions of the standard protein so as to ensure accurate estimation of your protein. In literature they used micro bca assay to determine liposome associated proteins. Different intensity present in reference protein even though bca performed reply. The bicinchoninic acid bca assay for protein quantitation.
Studies of the bca assay for determination of total. Micro bca protein assay reagent kit, sufficient reagents for 480 tube assays. Available in tube and microplate formats, these reducing agent compatible rac bca kits extend the already broad reagent compatibility of the bca protein assay protocol to include nearly all types of components commonly present in protein research samples. Pierce microplate bca protein assay kit reducing agent. The two assays are of similar sensitivity, but since bca is stable under alkali conditions, this assay has the advantage that it. Microsoft word ab102536 bca protein quantification kit protocol. The assay is linear over the widest range of protein concentration between 252000 gml. Lower protein samples can be more easily quantified using higher temperatures and longer incubation times. 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. The assay can be completed in 5 minutes at room temperature eliminating the need to wait or expose samples to elevated temperatures. Add 25 l of each bsa standard and protein samples into microtiter plate wells. I want to do a bca protein microplate assay pierce but our plate reader only has a 595 nm filter. Mix until the bca working reagent is a uniform, light green color. The two assays are of similar sensitivity, but since bca is stable under alkali conditions, this.