Bio 111 Lab 2 - Standard Curve and Dilutions

vocab + formulas

Standard Curve

A reference graph in which properties of known samples (BSA) are plotted so their direct proportionality can be used to calculate unknown sample concentrations.

Bovine Serum Albumin (BSA)

A low-cost, non-reactive blood protein derived from cows used as a reference concentration standard.

Bradford Assay

A colorimetric assay that quantifies protein concentration based on color change when protein binds dye reagent.

Coomassie Blue G-250

A dye reagent in the Bradford Assay that binds basic and aromatic amino acid side chains to form a blue protein-dye complex.

Bradford Assay Maximum Wavelength

595 nm (absorbance peak for the blue Coomassie protein-dye complex).

Spectrophotometer

An instrument that measures how much light a solution absorbs, which is directly proportional to solute concentration.

Spectrophotometer Light Path

Light Source -> Entrance Slit -> Dispersion Device (Prism) -> Exit Slit -> Sample Cuvette -> Detector.

Absorbance

The amount of light for a specific wavelength blocked or absorbed by a sample.

Optical Density (OD)

A logarithmic base-10 measure of absorbance (D = log10(I0/I)) reported in nanometers (nm).

Transmittance

The amount of light at a specific wavelength that travels completely through a sample.

Linear Trendline Equation

y = mx + b, where y is Absorbance (OD at 595 nm) and x is Protein Concentration.

Cuvette Concentration Equation (x)

x = (y - b) / m (or x = Absorbance / slope when y-intercept b = 0).

Table 1 BSA Unit Alignment

Convert 0.5 mg/mL BSA stock to 500 ug/mL by multiplying by 1000 before running C1V1 = C2V2.

Table 1 BSA Stock Volume Formula

V1 = (Target Conc. in ug/mL * 1 mL) / 500 ug/mL.

Table 1 Solution Balancing (NaCl Volume)

NaCl Volume = 1.0 mL Total - 0.9 mL Bradford Reagent - V1 BSA Stock Volume.

Table 3 Unknown Concentration Step 1

Calculate cuvette concentration (ug/mL) by dividing sample absorbance by the standard curve slope (x = Abs / m).

Table 3 Unknown Concentration Step 2

Convert unknown volume from uL to mL (divide by 1000) and solve for concentration in unknown volume via C1V1 = C2V2.

Table 3 Unknown Concentration Step 3

Multiply by the sample dilution factor to account for initial pre-dilution.

Table 3 Unknown Concentration Step 4

Convert final concentration from ug/mL to original units of ug/uL by dividing by 1000.

Milk Protein Assay Pre-Dilution

Milk samples are pre-diluted 1:50 in phosphate buffered saline before adding protein dye reagent.

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