Standard curve
Encyclopedia
A standard curve is a quantitative research
tool, a method of plotting assay
data that is used to determine the concentration
of a substance, particularly protein
s and DNA
. It can be used in many biological experiments
The assay is first performed with various known concentrations of a substance similar to that being measured. For example a standard curve for protein concentration is often created using known concentrations of bovine serum albumin
. The assay procedure may measure absorbance, optical density, luminescence
, fluorescence
, radioactivity, or something else.
Coomassie Brilliant Blue turns blue when it binds to arginine
and aromatic amino acid
s present in protein. The intensity of the colour is best measured at 595 nm, which is the maximum absorbance (Amax) frequency of the blue dye, using a spectrophotometer. In this case, the greater the absorbance, the higher the protein concentration.
These data are used to make the standard curve, plotting concentration on the X axis, and assay measurement on the Y axis. The same assay is then performed with samples of unknown concentration. To analyze the data, one locates the measurement on the Y-axis that corresponds to the assay measurement of the unknown substance and follows a line to intersect the standard curve. The corresponding value on the X-axis is the concentration of substance in the unknown sample.
Quantitative research
In the social sciences, quantitative research refers to the systematic empirical investigation of social phenomena via statistical, mathematical or computational techniques. The objective of quantitative research is to develop and employ mathematical models, theories and/or hypotheses pertaining to...
tool, a method of plotting assay
Assay
An assay is a procedure in molecular biology for testing or measuring the activity of a drug or biochemical in an organism or organic sample. A quantitative assay may also measure the amount of a substance in a sample. Bioassays and immunoassays are among the many varieties of specialized...
data that is used to determine the concentration
Concentration
In chemistry, concentration is defined as the abundance of a constituent divided by the total volume of a mixture. Four types can be distinguished: mass concentration, molar concentration, number concentration, and volume concentration...
of a substance, particularly protein
Protein
Proteins are biochemical compounds consisting of one or more polypeptides typically folded into a globular or fibrous form, facilitating a biological function. A polypeptide is a single linear polymer chain of amino acids bonded together by peptide bonds between the carboxyl and amino groups of...
s and DNA
DNA
Deoxyribonucleic acid is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms . The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in...
. It can be used in many biological experiments
The assay is first performed with various known concentrations of a substance similar to that being measured. For example a standard curve for protein concentration is often created using known concentrations of bovine serum albumin
Bovine serum albumin
Bovine serum albumin is a serum albumin protein derived from cows. It is often used as a protein concentration standard....
. The assay procedure may measure absorbance, optical density, luminescence
Luminescence
Luminescence is emission of light by a substance not resulting from heat; it is thus a form of cold body radiation. It can be caused by chemical reactions, electrical energy, subatomic motions, or stress on a crystal. This distinguishes luminescence from incandescence, which is light emitted by a...
, fluorescence
Fluorescence
Fluorescence is the emission of light by a substance that has absorbed light or other electromagnetic radiation of a different wavelength. It is a form of luminescence. In most cases, emitted light has a longer wavelength, and therefore lower energy, than the absorbed radiation...
, radioactivity, or something else.
Example
One assay for protein is called the Bradford assay; it is a colorimetric assay. The reagentReagent
A reagent is a "substance or compound that is added to a system in order to bring about a chemical reaction, or added to see if a reaction occurs." Although the terms reactant and reagent are often used interchangeably, a reactant is less specifically a "substance that is consumed in the course of...
Coomassie Brilliant Blue turns blue when it binds to arginine
Arginine
Arginine is an α-amino acid. The L-form is one of the 20 most common natural amino acids. At the level of molecular genetics, in the structure of the messenger ribonucleic acid mRNA, CGU, CGC, CGA, CGG, AGA, and AGG, are the triplets of nucleotide bases or codons that codify for arginine during...
and aromatic amino acid
Amino acid
Amino acids are molecules containing an amine group, a carboxylic acid group and a side-chain that varies between different amino acids. The key elements of an amino acid are carbon, hydrogen, oxygen, and nitrogen...
s present in protein. The intensity of the colour is best measured at 595 nm, which is the maximum absorbance (Amax) frequency of the blue dye, using a spectrophotometer. In this case, the greater the absorbance, the higher the protein concentration.
These data are used to make the standard curve, plotting concentration on the X axis, and assay measurement on the Y axis. The same assay is then performed with samples of unknown concentration. To analyze the data, one locates the measurement on the Y-axis that corresponds to the assay measurement of the unknown substance and follows a line to intersect the standard curve. The corresponding value on the X-axis is the concentration of substance in the unknown sample.
See also
- ColorColorColor or colour is the visual perceptual property corresponding in humans to the categories called red, green, blue and others. Color derives from the spectrum of light interacting in the eye with the spectral sensitivities of the light receptors...
- Linear regressionLinear regressionIn statistics, linear regression is an approach to modeling the relationship between a scalar variable y and one or more explanatory variables denoted X. The case of one explanatory variable is called simple regression...
- Logarithmic scaleLogarithmic scaleA logarithmic scale is a scale of measurement using the logarithm of a physical quantity instead of the quantity itself.A simple example is a chart whose vertical axis increments are labeled 1, 10, 100, 1000, instead of 1, 2, 3, 4...
- ProteinProteinProteins are biochemical compounds consisting of one or more polypeptides typically folded into a globular or fibrous form, facilitating a biological function. A polypeptide is a single linear polymer chain of amino acids bonded together by peptide bonds between the carboxyl and amino groups of...
- Serial dilutionSerial dilutionA serial dilution is the stepwise dilution of a substance in solution. Usually the dilution factor at each step is constant, resulting in a geometric progression of the concentration in a logarithmic fashion. A ten-fold serial dilution could be 1 M, 0.1 M, 0.01 M, 0.001 M.....