Each assay was performed in duplicate, and the results are presented as the average of duplicates. I had similar problems with the microplate so i switched to what they call the test tube procedure, or standard protocol. Download our protein assays and bioassays handbooks. The reaction reaches 90% of its maximum color development within 15 minutes thereby. To proceed dilute the contents of a 1 ml bsa standard 2 mgml into several clean vials, preferably using the same diluent as the test samples. Download fulltext pdf sulforhodamine b colorimetric assay for cytoxicity screening article pdf available in nature protocol.
Dilution scheme for standard test tube protocol and microplate procedure. The bca protein assay was performed following the manufacturers protocol. Studies of the bca assay for determination of total. Thermo scientific pierce protein assay technical handbook. The optimum measurement wavelength for this assay is 562 nm. The remaining kit components should be stored at room temperature. The absorbance at 562nm was read in a spectramax 250 plate reader. The bicinchoninic acid bca protein assay is a highly sensitive colorimetric assay that is compatible with. Compatibility of pierce bca protein assay with promega lysis. Bca protein assay with protein concentration curve standard. Linear regression curve fit is used to determine unknown concentrations. The bicinchoninic acid bca protein assay is a highly sensitive colorimetric assay that is compatible with detergent solubilized protein solutions.
Competitive binding to cuprous ions of protein and bca in. A purple colored reaction product is formed by the chelation of two molecules of bca with one cuprous ion. Bicinchonininsaure bca einen violetten komplex bilden. Bicinchoninic acid protein assay kit bca1 technical bulletin. The enhanced protocol requires a 30minute incubate at 60c. Discard any kit reagent that shows discoloration or evidence of microbial contamination. Plate map with a standard curve from 0 to 100 gml with samples and standards in duplicate. The assay is linear over the widest range of protein concentration between 252000 gml. Bca enhanced protocol assay was performed on the modulus using the. The bicinchoninic acid bca assay, first described by smith et al.
I used bsa provided in the kit as a standard and the correlation coefficient was very good 0. The biorad dc protein assay is a colorimetric assay for protein concentration following detergent solubilization. Biovisions reducing agent compatible bca protein assay kit is the only protein estimation kit on the market that is compatible with. Protein concentrations between 5250 gml can be determined with this protocol. Coomassie dyebased protein assays bradford assays 19. Mar 27, 2020 download a modulus method for bca protein assay book pdf free download link or read online here in pdf. Improved file size, sorting and search tools for working with gen5s database and sample file database integrates with all biotek detection systems gen5 microplate reader and imaging software is an integrated tool for data collection and analysis, compatible with all of bioteks multimode and single mode readers. Bca versus biorad protein assay what are the advantages of bca that all the world uses it. Bca protein assay with protein concentration curve. If you treat them the same way as the samples, that is by adding the sds. 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.
Incompatible substances amount compatible buffer systems nacetylglucosamine 10 mm in pbs, ph 7. Bca assay high levels of ionic detergents require the addition of the ionic detergent compatibility reagent idcr. Measurement of protein using bicinchoninic acid sciencedirect. I recently migrated from the bradford method to the microbca method from pierce for the quantification of total protein from cell culture. A modulus method for bca protein assay pdf book manual.
The bicinchoninic acid bca assay for protein quantitation. The biorad protein assay is a simple colorimetric assay for measuring total protein concentration and is based on the bradford dyebinding method bradford 1976. The enhanced protocol requires a 60c incubation for 30 minutes. The bca assay is used to determine the concentration of peptide bonds in a protein sample quantitatively. Bca protein assay kit reducing agent compatible rev. Bicinchoninic acid bca assay in low volume sciencedirect. The standards used for the bca assay usually bovine serum albumin should contain the same amount of sds as the samples. Biotek sample files, 08jun11, bca protein assay with protein concentration curve standard curve and calculation of unknown concentrations. More sensitive than lowry and less subject to interferences. The bicinchoninic acid bca protein assay is based on a twostep reaction in one tube. Bsa bovine serum albumin is used as the standard and samples are. Be aware of any hazardous compounds, take precautions where necessary and dispose of any waste in the appropriate manner. The first step is the chelation of copper with protein in an alkaline environment to form a light blue complex. Bca protein assay photometric method reading at 562 nm.
The two assays are of similar sensitivity, but since bca is stable under alkali conditions, this assay has the advantage that it can. The bca protein assay, also known as the smith assay, is a highly sensitive colorimetric assay that is compatible with detergent solubilized protein solutions and primarily relies on two reactions. 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. 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. Certain substances interfere with the pierce bca assay kit. Download a modulus method for bca protein assay book pdf free download link or read online here in pdf. Pierce bca protein assay kit, sufficient reagents for 500 testtube or 5000. The following tables provide information to prepare a set of protein standards for a standard curve for common bca assay and bradford assays. The bicinchoninic acid bca 2 assay is a colorimetric method that is commonly employed to estimate the concentration of protein in a sample. The protocols described here are for guidance only.
Bicinchoninic acid protein assay kit bca1 technical. Be sure to update the file to specify the location of the standards, blanks, and unknowns before reading 6. 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. Protein assay data analysis thermo fisher scientific ng. The bicinchoninic acid bca protein assay primarily relies on two reactions. In test tubes or 96well plates, the relationship between protein concentration and absorbance is nearly linear over a. Bca protein assay for estimating detergent solubilized proteins.
These and others changes like the bca working reagent to protein sample ratio affect the net absorbance values. The bca protein assay reducing agent compatible standard protocol. The standard protocol permits either a 30minute incubation at 37c or a 2hour incubation at room temperature. For more product information, or to download a product instruction booklet, visit. The bca assay is the best indirect type of protein assay for protein to protein measurement. Competitive binding to cuprous ions of protein and bca in the. 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. Compatibility of pierce bca protein assay with promega. This site is like a library, you could find million book here. 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. The takara bca protein assay kit is a highly sensitive colorimetric assay that is compatible with detergent solubilized protein solutions. This was done by analyzing the precision, accuracy, linearity and range of the method. Studies of the bca assay for determination of total protein.
Pdf the bca assay is a colorimetric method for estimating protein concentration. Pdf bicinchoninic acid bca assay in low volume researchgate. By modifying the protocol you can change the range to 202000 gml. To minimize the affect of these copper reducers, a compatibility reagent that. 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. Microplate bca assay for wholecell lysate entered by. 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. Protein assay compatibility table from our website. Bca protein assay for estimating detergent solubilized. The bicinchoninic acid assay bca assay, also known as the smith assay, after its inventor, paul k. Microplate bca assay for wholecell lysate entered by kevin janes janes lab protocols 31209 1 1.
This product is guaranteed for one year from the date of purchase when. I have found the assay to be fairly sensitive and reliable, however a few points should be noted. The assay can be completed in 5 minutes at room temperature eliminating the need to wait or expose samples to elevated temperatures. Pierce bca protein assay kit thermo fisher scientific. The two assays are of similar sensitivity, but since bca is stable under alkali conditions, this. Add 10l of bsa standards stored in 80c freezer to each of the appropriate wells. Protein assay lab report using the bca assay to determine. In particular, it is insensitive to detergents such as triton x100 and sds 1 %. Module and the bca protein assay is 5 gml figure 1 and the maximum is 2 mgml figure 2. A 25l sample volume is recommended for the microplate procedure of the bca protein assay. The two assays are of similar sensitivity, but since bca is stable under alkali conditions, this assay has the advantage that it. Introduction beers law tells us that the light absorbance a of a solution is. The pierce bca protein assay is a detergentcompatible formulation based on bicinchoninic acid bca for the colorimetric detection and quantitation of total protein.
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. The reaction is similar to the welldocumented lowry1 assay, but with the following improvements. 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. A typical bca assay standard curve with samples ranging from 0. The bca protein assay is used for quantitation of total protein in a sample. This curve is then utilized to determine the concentration of protein in an unknown solution. This is the protocol to be used in the determination of total protein in brain tissue by the pierce bca method. Dc protein assay instruction manual hebrew university of. The pathlength in each assay is dependent on the assay container cuvettes or multiwell plates andor the reaction volume. Add 10l of unknown sample to each of the appropriate wells. Read online a modulus method for bca protein assay book pdf free download link book now.
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