Podcast
Questions and Answers
What does isopycnic centrifugation primarily separate based on?
What does isopycnic centrifugation primarily separate based on?
Which substance is commonly used to create a density gradient in isopycnic centrifugation?
Which substance is commonly used to create a density gradient in isopycnic centrifugation?
How does rate zonal centrifugation differ from isopycnic centrifugation?
How does rate zonal centrifugation differ from isopycnic centrifugation?
In molecular exclusion chromatography, what defines the 'excluded volume'?
In molecular exclusion chromatography, what defines the 'excluded volume'?
Signup and view all the answers
What happens to very large size protein molecules during molecular exclusion chromatography?
What happens to very large size protein molecules during molecular exclusion chromatography?
Signup and view all the answers
What is the purpose of washing and equilibrating the gel material in molecular exclusion chromatography?
What is the purpose of washing and equilibrating the gel material in molecular exclusion chromatography?
Signup and view all the answers
What is the primary measurement unit for buoyant density?
What is the primary measurement unit for buoyant density?
Signup and view all the answers
Why are small proteins retarded in a molecular exclusion chromatography column?
Why are small proteins retarded in a molecular exclusion chromatography column?
Signup and view all the answers
During which phase do submicroscopic protein aggregates begin to form?
During which phase do submicroscopic protein aggregates begin to form?
Signup and view all the answers
What initiates the protein precipitation process?
What initiates the protein precipitation process?
Signup and view all the answers
What largely influences the concentration of salt needed for protein precipitation?
What largely influences the concentration of salt needed for protein precipitation?
Signup and view all the answers
What happens to protein solubility as salt concentration increases?
What happens to protein solubility as salt concentration increases?
Signup and view all the answers
In differential centrifugation, how are organelles primarily separated?
In differential centrifugation, how are organelles primarily separated?
Signup and view all the answers
What is the result of the dehydration of proteins during the salting-out process?
What is the result of the dehydration of proteins during the salting-out process?
Signup and view all the answers
What is the primary purpose of centrifugation in biological studies?
What is the primary purpose of centrifugation in biological studies?
Signup and view all the answers
Which of the following is NOT a substance mentioned that can be used to create a density gradient?
Which of the following is NOT a substance mentioned that can be used to create a density gradient?
Signup and view all the answers
What is the purpose of lysing the bacteria in the protein isolation process?
What is the purpose of lysing the bacteria in the protein isolation process?
Signup and view all the answers
What is one consequence of continuing to centrifuge a sample beyond the necessary time?
What is one consequence of continuing to centrifuge a sample beyond the necessary time?
Signup and view all the answers
What happens to organelles during velocity centrifugation?
What happens to organelles during velocity centrifugation?
Signup and view all the answers
At what critical size do protein particles stop growing through collision and flocculation?
At what critical size do protein particles stop growing through collision and flocculation?
Signup and view all the answers
What role does the density gradient play in density gradient centrifugation?
What role does the density gradient play in density gradient centrifugation?
Signup and view all the answers
How does the activity of the protein of interest change with increasing concentrations in the supernatant?
How does the activity of the protein of interest change with increasing concentrations in the supernatant?
Signup and view all the answers
Which centrifugation technique separates organelles primarily based on their density?
Which centrifugation technique separates organelles primarily based on their density?
Signup and view all the answers
Which is true regarding the order of separation in differential centrifugation?
Which is true regarding the order of separation in differential centrifugation?
Signup and view all the answers
What is the primary mechanism by which moving-boundary electrophoresis separates compounds?
What is the primary mechanism by which moving-boundary electrophoresis separates compounds?
Signup and view all the answers
What measurement is used to detect the movement of compounds in moving-boundary electrophoresis?
What measurement is used to detect the movement of compounds in moving-boundary electrophoresis?
Signup and view all the answers
What is the purpose of a purification table in enzyme purification?
What is the purpose of a purification table in enzyme purification?
Signup and view all the answers
How is the isoelectric pH of a protein typically determined using moving-boundary electrophoresis?
How is the isoelectric pH of a protein typically determined using moving-boundary electrophoresis?
Signup and view all the answers
What components are included in the moving-boundary electrophoresis apparatus?
What components are included in the moving-boundary electrophoresis apparatus?
Signup and view all the answers
Which type of chromatography is based on specific binding properties?
Which type of chromatography is based on specific binding properties?
Signup and view all the answers
What does the degree of purification in an enzyme purification process indicate?
What does the degree of purification in an enzyme purification process indicate?
Signup and view all the answers
In moving-boundary electrophoresis, what is the direction of compound migration based on?
In moving-boundary electrophoresis, what is the direction of compound migration based on?
Signup and view all the answers
What is the primary purpose of the low ionic strength buffer used in ion exchange chromatography?
What is the primary purpose of the low ionic strength buffer used in ion exchange chromatography?
Signup and view all the answers
In ion exchange chromatography, what happens to proteins that are charged oppositely to the ion-exchange media during the application step?
In ion exchange chromatography, what happens to proteins that are charged oppositely to the ion-exchange media during the application step?
Signup and view all the answers
Which method can be used to elute proteins from the column aside from increasing ionic strength?
Which method can be used to elute proteins from the column aside from increasing ionic strength?
Signup and view all the answers
What is the main difference between adsorption chromatography and ion exchange chromatography?
What is the main difference between adsorption chromatography and ion exchange chromatography?
Signup and view all the answers
In adsorption chromatography, how does the binding strength to the stationary phase affect the movement of molecules?
In adsorption chromatography, how does the binding strength to the stationary phase affect the movement of molecules?
Signup and view all the answers
Which material is commonly used as a stationary phase in adsorption chromatography?
Which material is commonly used as a stationary phase in adsorption chromatography?
Signup and view all the answers
What characteristic of silica makes it suitable for use as a stationary phase in chromatography?
What characteristic of silica makes it suitable for use as a stationary phase in chromatography?
Signup and view all the answers
How does the migration rate of proteins in electrophoresis relate to their physical characteristics?
How does the migration rate of proteins in electrophoresis relate to their physical characteristics?
Signup and view all the answers
Study Notes
Centrifugation Techniques
- Centrifugation isolates pure samples of cell organelles for biochemical study.
- Differential centrifugation separates organelles based on weight; heaviest first at low speeds, lighter at high speeds.
- Density gradient centrifugation separates organelles based on density using sucrose solutions layered in a test tube.
- Velocity (rate-zonal) centrifugation uses a sucrose gradient to create bands of components based on sedimentation speed without separating them by buoyancy.
- Equilibrium (isopycnic) centrifugation separates components by buoyant density, an equilibrium method where components remain stable once reached.
Molecular Exclusion Chromatography
- Uses hydrated polymer materials like dextran and agarose to prepare the column for separating proteins based on size.
- Larger proteins are excluded from entering pores, moving faster through the column; smaller ones diffuse into the beads and travel slower.
- Initial mixing involves adding a precipitating agent, leading to nucleation and aggregation of protein particles until a stable size is reached.
- Salting out relies on salt concentration to precipitate proteins by reducing solubility and encouraging self-association.
Protein Isolation Process
- For isolating a novel protein: bacteria cultured, harvested by centrifugation, lysed using freeze/thaw, followed by centrifugation to collect soluble proteins.
- Assays measure biological activity with a color change correlated to protein concentration.
Ion Exchange Chromatography Steps
- Proteins are transferred into a low ionic strength buffer and applied on a pre-equilibrated column with the same buffer.
- Oppositely charged proteins are retained, while non-adhering proteins pass through.
- Bursts in ionic strength gradually elute retained proteins by increasing net positive or negative charges.
Adsorption Chromatography
- Characterized by separating components based on their adsorption to the stationary phase within the column.
- The efficiency of separation depends on solubility in the mobile phase and binding strength to the stationary phase (e.g., alumina or silica).
- Incorporates both positive and negative charges, affecting protein migration and separation.
Electrophoresis Techniques
- Electrophoresis separates proteins in an electric field, migration rate influenced by the system and protein characteristics.
- Moving-boundary electrophoresis uses a cell with buffer and electrodes to detect separation via refractive index changes at compound boundaries.
- Isoelectric focusing determines the isoelectric pH at which proteins have no net charge, aiding in their identification.
Affinity Chromatography
- Separates proteins based on specific binding properties; highly selective for target proteins.
Purification Tables
- Purification tables track enzyme purification progress, listing total activity, total protein, specific activity, fold purification, and percentage recovery.
- Each step indicates quantities to monitor yield and relative purity at each purification phase.
Example Enzyme Purification
- Calculating specific activity, percent yield, and degree of purification for an enzyme like acid phosphatase helps identify the most effective purification step based on fold increase.
Studying That Suits You
Use AI to generate personalized quizzes and flashcards to suit your learning preferences.
Description
This quiz covers essential techniques in centrifugation and molecular exclusion chromatography. Learn how differential, density gradient, velocity, and equilibrium centrifugation methods isolate cell organelles, as well as the principles behind size-based protein separation in chromatography. Test your knowledge on these critical laboratory methods.
