flashcard 13
(50 cards)
What distinguishes protein chromatography from small-molecule chromatography?
Protein chromatography often requires additional sample preparation and purification steps to isolate and maintain native protein function, whereas small-molecule chromatography typically handles simpler mixtures without extensive biological matrix cleanup.
Why is sample preparation critical before protein chromatography?
Because proteins are extracted from complex biological materials, steps like cell/tissue lysis, precipitation, centrifugation, and dialysis remove unwanted debris, concentrate the protein of interest, and stabilize it to prevent degradation during chromatography.
How does homogenization contribute to protein extraction?
Homogenization mechanically disrupts cell or tissue structure, breaking open membranes to release intracellular proteins into solution.
What role does sonication play in protein purification?
Sonication uses high-frequency sound waves to lyse cells by creating cavitation, releasing intracellular proteins while often preserving protein activity if conditions are controlled.
Why might freeze–thaw cycles be used in protein extraction, and what is a drawback?
Repeated freezing and thawing disrupts cell membranes by ice crystal formation, releasing proteins; however, it can also denature some proteins and is time-consuming.
How do detergents assist in protein purification?
Detergents solubilize membrane components, keeping proteins in solution; mild detergents preserve native structure, while stronger ones like SDS denature proteins to enable downstream analysis.
What is ‘salting out,’ and which reagent is commonly used?
Salting out precipitates proteins by reducing their solubility at high ionic strength; ammonium sulfate is frequently used to selectively precipitate and fractionate proteins.
How does differential centrifugation fractionate cellular components?
By spinning lysate at progressively higher speeds, larger/heavier components pellet first, leaving soluble proteins in the supernatant for further purification.
What is the purpose of dialysis in protein purification?
Dialysis removes small contaminants by diffusion through a semipermeable membrane, allowing separation of high–molecular-weight proteins from low–molecular-weight impurities.
Why is stabilizing proteins important during purification?
Many proteins are sensitive to temperature, pH, or proteases; adding stabilizing agents and maintaining optimal buffers prevent denaturation and loss of activity.
What does the term ‘polishing’ refer to in protein purification?
Polishing is the final purification step, achieving the highest purity by removing remaining contaminants, often using high-resolution chromatography or additional selective methods.
How does column equilibration prepare for protein binding?
Equilibration flushes the column with the desired buffer conditions to establish a stable environment ensuring reproducible binding interactions when the protein sample is applied.
In ion exchange chromatography, what defines a cation exchanger versus an anion exchanger?
A cation exchanger has negatively charged groups on the resin to bind positively charged proteins; an anion exchanger has positively charged groups to bind negatively charged proteins.
How does a protein’s isoelectric point (pI) influence its behavior in ion exchange chromatography?
At a pH below its pI, a protein carries a net positive charge and will bind to a cation exchanger; above its pI, it becomes net negative and will bind to an anion exchanger, or elute if the opposite resin is used.
What is gradient elution in ion exchange chromatography, and why is it used?
Gradient elution gradually increases salt concentration or alters pH in the mobile phase to weaken ionic interactions, sequentially eluting bound proteins according to their binding strength.
Given two peptides—Peptide A (pI 5.1, net negative at pH 7) and Peptide B (pI 7.8, net positive at pH 7)—which elutes first from a cation exchange column at pH 7?
Peptide A elutes first because it is net negative at pH 7 and does not bind strongly to the negatively charged resin, whereas Peptide B is net positive and binds tightly.
In the same scenario, which peptide would elute first from an anion exchange column at pH 7?
Peptide B elutes first from an anion exchanger because it is net positive at pH 7 and does not bind to the positively charged resin, while Peptide A (net negative) binds tightly and elutes later.
What is size exclusion chromatography (SEC), and how does it separate proteins?
SEC separates molecules by size: large proteins bypass resin pores and elute earlier, while smaller proteins enter pores, are retarded, and elute later, effectively fractionating by hydrodynamic volume.
Define the void volume (V₀) in size exclusion chromatography.
Void volume is the volume of mobile phase within the column that excludes all pores—molecules too large to enter any pores elute at this volume.
What is the exclusion limit in a size exclusion column?
The exclusion limit is the minimum molecular size above which molecules cannot enter any pores and thus elute at the void volume.
What is the inclusion limit in size exclusion chromatography?
The inclusion limit is the maximum molecular size that can fully access all pores; such small molecules elute at the total column volume after exploring the entire pore network.
How can one estimate a protein’s molecular weight using SEC?
By running molecular weight standards to create a calibration curve of log(MW) versus retention volume, then plotting the unknown protein’s retention volume to interpolate its molecular weight.
What is affinity chromatography, and what key components does it require?
Affinity chromatography uses a resin with immobilized ligand specific to the target protein; key components include the matrix, a spacer arm, and the reversible ligand that binds the protein.
Why is a spacer arm used in affinity chromatography?
A spacer arm distances the ligand from the resin surface, minimizing steric hindrance and allowing the target protein to bind more efficiently to the ligand.
