Unit 4 - Determining Protein Concentration

Vocabulary flashcards covering protein properties, amylase, Bradford reagent mechanism, spectrophotometry components, absorbance/transmittance relationships, and standard curve calibration.

Protein

A complex macromolecule made up of amino acids that possesses 4 levels of structure and performs a wide range of functions including structural, transport, signaling, and catalytic roles.

Amylase

A catalytic protein (enzyme) produced by the pancreas and salivary glands that catalyzes the breakdown of starch into smaller carbohydrates.

Bradford Reagent

A protein concentration assay reagent based on Coomassie brilliant blue G-250 dye and copper, which reacts with arginine, lysine, and histidine amino acids to produce a blue color.

Coomassie brilliant blue G-250

The dye component of Bradford reagent that binds to protein amino acids, shifting its absorbance maximum to $595\,nm$ to produce a blue-colored solution.

Spectrophotometer

An instrument used to measure the absorbance and transmittance of light through a solution to determine sample concentrations.

Spectrophotometer Components

An optical measuring system comprising a Light source, Collimator (lens), Monochromator (prism or grating), Wavelength selector (slit), Sample solution (in cuvette), Detector (photocell), and Digital display or meter.

Spectronic 200 Control Panel

The user interface on the spectrophotometer consisting of a Wavelength Knob, Zero button ($0.00$), Home Button, and Enter Button used for setting wavelengths and calibrating absorbance.

Transmittance (%T)

The percentage of light passing through a solution that reaches the detector; values of percent transmittance $\%T < 10\%$ are considered unreliable when using Bradford reagent.

Absorbance

The measure of light absorbed by a solution, calculated as the negative logarithm of transmittance ($A = -\log(\%T)$), where values $A > 1$ are considered unreliable using Bradford reagent.

Blank ("B" Tube)

A reference sample containing di$\text{H}_2\text{O}$ and Bradford reagent without protein, used to calibrate ("zero") the spectrophotometer before measuring experimental samples.

Standard Curve

A linear graph created by plotting absorbance against known protein concentrations, using a line of best fit ($y = mx + b$) to estimate unknown protein concentrations.

Serial Dilution

A systematic, stepwise dilution of a standard protein solution used to prepare a series of known concentrations for generating a standard curve.