Week 5: Protein Techniques 1 Flashcards
What technique is used for the determination of protein concentration?
Spectrophotometry
Spectrophotometry is important for the determination of concentration of a wide range of biomolecules including proteins, nucleic acids, and metabolites.
What is the purpose of differential centrifugation?
Isolation of organelles
Isolation of organelles can simplify protein purification.
Name a method for separating proteins based on size.
Gel-filtration chromatography
Gel-filtration chromatography separates proteins on the basis of size.
What are the key characteristics used to characterize a protein?
- Size
- Protein sequence
- Structure
- Activity
These characteristics help in the purification and analysis of proteins.
Where can proteins be sourced from for isolation?
- Tissue or organ
- One type of cell in culture
- Microorganisms (e.g., bacteria and yeast)
Microorganisms can be engineered using recombinant DNA technology to produce large amounts of a particular protein.
What forms of electromagnetic radiation are involved in spectrophotometry?
- Visible light
- Ultraviolet radiation
- X-rays
Electromagnetic radiation travels in waves and differs in wavelengths and energy.
True or False: The absorbance of light is the amount of radiation transmitted.
False
Absorbance is the amount of radiation absorbed, not transmitted.
What is the role of a homogenizer in protein extraction?
To break up cells
A glass-Teflon homogenizer or blender is used to create a homogenate or crude extract.
What is salting out in protein purification?
Precipitating proteins at different salt concentrations
Salting out takes advantage of differences in solubility among proteins.
What chromatography technique separates proteins based on charge?
Ion-exchange chromatography
This technique utilizes differences in the charge of proteins for separation.
Fill in the blank: _______ chromatography separates proteins based on specific binding affinities.
Affinity
Some proteins have a high affinity for specific chemicals or chemical groups.
What is High-Performance Liquid Chromatography (HPLC) known for?
Greater resolving power than normal columns
HPLC uses fine beads and high-pressure pumps to move liquids through the column.
What is the first step in determining protein structure by X-ray crystallography?
Preparing a protein crystal
This involves exposing the crystal to X-rays and detecting scattered X-rays.
What is cryo-electron microscopy used for?
Determining protein structure
It allows for the visualization of protein complexes at near-atomic resolution.
What is the general summary of protein techniques discussed?
- Determination of biomolecule concentration by spectrophotometry
- Isolation of organelles by differential centrifugation
- Protein separation using chromatography based on size, charge, and binding affinity
- Determination of protein structure by X-ray crystallography and cryo-EM
These techniques are essential for understanding protein function and characteristics.
