Protein Study Techniques Flashcards
What are the three steps for extracting cellular proteins?
- Cell Rupture (Homogenization)
- Differential Centrifugation
- Purifying Soluble Proteins
What happens during the Cell Rupture (Homogenization) stage? Why is it important?
It’s just a step that breaks open the cell and releases its contents into a solution.
This step is critical for accessing intracellular proteins while maintaining their structure and function as much as possible.
What are the steps of Differential Centrifugation? (Add speeds)
Three types of spins:
Low speed: Pellet includes nuclei and large debris (Largest) ~600g
Medium speed: Pellet includes mitochondria and medium-sized debris ~15000g
High Speed: Pellet includes microsomes and ribosomes (Smallest) ~100000g
What is “salting out”?
It’s a way of partially purifying proteins by selectively precipitating them out of solution.
How does salting out work?
It creates a situation where salt competes with proteins for water molecules thus decreasing the solubility of the proteins precipitating them out of the solution. (start with a low concentration then slowly increase salt concentration) (Hydrophobic proteins will precipitate first)
What are the steps of Protein Purification?
Salt Fractionation (Salting Out): Precipitate specific proteins using salt.
Dialysis: Remove small molecules and salts from the protein solution.
Chromatography: A method to separate proteins (Based on charge, size, etc)
Electrophoresis: Final analysis and potential additional purification step.
What is the difference between total activity and specific activity?
Total activity: The total enzymatic activity or functional protein present in the sample. It is a product of the amount of protein and its catalytic rate.
Specific activity: The ratio of enzymatic activity (or protein function) to the amount of total protein. It’s a measure of purity and effectiveness.
How does total activity and specific activity change during the purification process?
During the early steps: Total activity declines and specific activity remains low
During the intermediate steps: Total activity declines but is slower and specific activity starts to increase
During the final steps: Total activity may decrease and specific activity reaches its peak
What are the two phases of chromatography? What is their purpose?
Stationary Phase: The stationary phase is the immobile phase of the chromatographic system where the separation of molecules occurs.
Mobile Phase: The mobile phase is the solvent or buffer that flows through the stationary phase, causing the molecules to be separated.
What is the column chromatography method?
In column chromatography, the stationary phase is packed in a column, and the mobile phase (eluent) moves through it, allowing for separation based on interactions with the stationary phase.
What is the size exclusion (gel filtration) chromatography method?
In size exclusion chromatography, separation is based on molecular size, with large molecules eluting before small ones due to the size-selective pores in the stationary phase.
What is affinity chromatography and what are the idk?
Affinity chromatography leverages specific interactions between proteins and ligands for high-purity purification.
What is ion exchange chromatography and what are the idk?
Ion exchange separates proteins based on charge, with the resin binding proteins of the opposite charge. Proteins are released when pH equals their isoelectric point and thus lose charge.
What is HPLC and what is its purpose?
HPLC (or High-Pressure Liquid Chromatography) offers faster and cleaner separation due to high pressure and precise control of the stationary phase.
How do things separate via electrophoresis?
Electrophoresis separates proteins based on their charge, shape, and size when they are placed in an electric field. (THINGS RUN FROM THE NEGATIVE TERMINAL TO THE POSITIVE ONE)
