Lab 5 Protein Analysis

Cell Fractionation Process

Cell Fractionation is a technique used to separate different components of a cell (organelles, proteins, etc.) based on size, density, or other properties.

• Step-by-Step Process:

  1. Homogenization: The cell membrane is broken open (typically by using a homogenizer or blender), releasing the cell contents into a buffer solution.

  2. Centrifugation: The homogenate (mixture of cell contents) is centrifuged at increasing speeds.

     • Low speed: Nuclei and large organelles (like the mitochondria) pellet first.

     • Medium speed: Smaller organelles (like lysosomes and peroxisomes) are pelleted.

     • High speed: Ribosomes, proteins, and other small molecules remain in the supernatant.

  3. Fractionation: The pellets and supernatants contain different parts of the cell, which can be analyzed separately.

  • Purpose: To isolate specific organelles for further study or experimentation.

Bradford Assay

The Bradford Assay is a colorimetric protein assay that measures protein concentration based on the binding of Coomassie Brilliant Blue dye to proteins.

• How It Works:

  • The dye (Coomassie Brilliant Blue) binds to basic and aromatic amino acid residues in proteins.

  • This binding causes a shift in the dye’s absorbance maximum from 465 nm (brown) to 595 nm (blue).

  • The intensity of the blue color is directly proportional to the protein concentration.

• Steps of the Bradford Assay:

  1. Add Bradford reagent to the sample.

  2. Incubate for a few minutes (typically 5-10 minutes).

  3. Measure the absorbance at 595 nm using a spectrophotometer.

Reading a Standard Curve

A Standard Curve is used to determine the concentration of an unknown sample based on known concentrations.

• How to Read It:

  1. Plot the Standard Curve:

     • On the x-axis: plot known protein concentrations (usually in micrograms per milliliter).

     • On the y-axis: plot the corresponding absorbance at 595 nm (measured from the Bradford assay).

  2. Generate the Line:

     • The data points are fitted to a straight line (usually using linear regression).

  3. Determine the Unknown Concentration:

     • Measure the absorbance of the unknown sample.

     • Use the standard curve to find the protein concentration corresponding to that absorbance value.

Protein Quantification Using Bradford Assay

Process:

• The concentration of protein in a sample can be determined by comparing its absorbance (after adding Bradford reagent) to the absorbances of known standards (from the standard curve).

• The protein concentration of the unknown sample can then be interpolated from the standard curve.

Basics of Spectrophotometry

Spectrophotometry is a method used to measure the amount of light that is absorbed by a sample.

• Principle:

  • Light of a specific wavelength passes through the sample.

  • The absorbance of light by the sample is measured, which is related to the concentration of a substance in the sample.

    BEER-LAMBERT LAW

A=ϵ⋅c⋅l

• A = Absorbance

• ε = Molar absorption coefficient (a constant for a particular substance at a specific wavelength)

• c = Concentration of the substance

• l = Path length of the sample (usually in cm)

• Uses:

  • Commonly used for determining concentrations of proteins, DNA, RNA, or other substances in solution.