BIT373 - Week 6 - Protein Quantification - Bradford

Bradford Assay

A method for protein quantification that uses Coomassie Brilliant Blue dye; the absorbance changes from 465nm to 595nm when proteins bind.

Standard Curve

A graph that correlates the concentration of a substance to its absorbance, used for quantifying unknown samples.

Coomassie Brilliant Blue

A dye used in the Bradford Assay that binds to proteins, particularly affecting aromatic and basic R-groups.

SimpliNano Spectrophotometer

A microvolume spectrophotometer that measures protein concentration at (280NM) and nucleic acid at (260nm) purity using UV light.

UV Spectrophotometry

A method of measuring light absorbance at specific wavelengths to determine protein concentration; affected by other molecules.

Protein Purity

The ratio of the desired protein (e.g., GFP) to the total protein present in a sample.

Dilution

The process of reducing the concentration of a solution by adding more solvent; used for preparing standards.

Aromatic R-groups

Side chains in amino acids that contain an aromatic ring, which can interact with certain dyes like Coomassie Brilliant Blue.

Sensitivity

The ability of a method to detect low concentrations of a substance; important in protein quantification methods.

Serial Dilution

A stepwise dilution of a substance where a solution of known concentration is diluted to create a series of lower concentrations.

Total Protein

The sum of all proteins present in a sample, necessary for calculating purity.

GFP/Total Protein ratio

A measurement for determining the purity of GFP in a sample relative to the total protein concentration.

Absorbance Measurement

A technique to measure how much light is absorbed by a sample, used in various assays for quantifying substances.

Advantages of Bradford Assay

Very sensitive so it has very little loss. know to be fast when binding as well as to revolve around limiting the sensitivity of the contaminates.

How does colorimetric Assay Work

They work by binding onto A crude Impure protein. This method Is successful Through the binding of a dye to a particular amino acid and peptide bond

How does the binding with a dye occurs

Known to operate through The sulfonic Group and the Amino acids of basic Charge And the Wan der Waals Interactions occurring Through the aromatic Hydrophobic Group.