Orla CA 1 Flashcards
What are the different types of chromatography based on the mobile and stationary phases?
Liquid-liquid: Phase separation using two liquids
Liquid-solid: Liquid mobile phase, solid stationary phase
Gas-solid: Gas mobile phase, solid stationary phase
Gas-liquid: Gas mobile phase, solid stationary phase coated with liquid molecules
What is column chromatography?
A separation method that uses a stationary phase and a mobile phase that flows over the stationary phase. Separation occurs based on the material’s interaction with the stationary phase relative to the mobile phase.
What are the differences between Partition and Adsorption Chromatography?
Partition = separation based on the distribution of solutes between two liquid phases
adsorption chromatography = separatiom based on solutes binding to a solid phase
What is required for the stationary phase in chromatography?
The stationary phase needs
Hydration
Inert matrix
reactive groups that interact with the sample’s components.
What is protein purification?
Extracting the total protein content from a source, followed by fractionation to isolate specific proteins.
How is cell bursting performed in protein extraction?
Cell bursting involves using buffers and stabilizing conditions (e.g., low temperatures, sonication, freeze-thaw) to extract proteins
How is a protein stabilized during purification?
By using buffers to maintain optimal pH and salt concentration, keeping heat-labile proteins on ice, and using protease inhibitors.
What is differential centrifugation?
It is a technique for separating cell components after cell bursting, allowing you to isolate specific fractions, which are then assayed to find the component of interest.
What are common concentration methods used in protein purification?
Salting out: High concentrations of ions (e.g., ammonium sulfate) bind water, causing proteins to precipitate.
Ultrafiltration: A membrane with specific molecular weight cut-off filters small components, retaining proteins.
Dialysis: Used to remove small molecular weight contaminants or salt before chromatography.
What determines the charge of a protein?
The charge depends on the sequence of amino acids and the isoelectric point (pI), which is the pH at which the overall charge of the protein is zero.
How can the charge of a protein be estimated?
When the pH > pI, the protein is negatively charged.
When the pH < pI, the protein is positively charged.
What are the main types of ion exchange chromatography?
Anion exchange chromatography: Positively charged medium binds negatively charged proteins.
Cation exchange chromatography: Negatively charged medium binds positively charged proteins
How can proteins be eluted from ion exchange columns?
By changing the pH of the buffer or increasing the salt concentration to disrupt the charge interaction between the protein and the column.
What is the difference between a strong and weak ion exchanger?
Strong ion exchanger: Maintains charge over a broad pH range; easy to develop/optimize but less selective.
Weak ion exchanger: Maintains charge over a narrow pH range; offers different selectivity but has longer equilibration times.
What is gel filtration chromatography?
It is a size-based separation method where proteins pass through pores in the gel matrix. Larger proteins elute first, while smaller proteins elute last.
What are the main purposes of gel filtration chromatography?
To remove low molecular weight components (e.g., desalting).
To determine the molecular mass of proteins.
To separate protein mixtures with varying molecular masses.
What are common gel filtration matrices?
Biogel: Polyacrylamide-based.
Superose: Cross-linked agarose.
Sepharose: Agarose-based.
How are flow rates determined in gel filtration chromatography?
Flow rates depend on column size and the experiment type (analytical, preparative, or large-scale purification).
What are key requirements for gel filtration?
The protein sample must be concentrated, and only one buffer should be used at an optimal pH for the protein of interest.
