flashcard 14
(50 cards)
What is the purpose of polyacrylamide gel electrophoresis (PAGE) in protein characterization?
PAGE separates proteins by acting as a molecular “sieve,” slowing their migration in proportion to their charge-to-mass ratio (and partially their shape), allowing assessment of purity and approximate molecular weight.
What two physical factors determine electrophoretic mobility (μ) in PAGE?
Electrophoretic mobility is the ratio of protein velocity (V) to the electric potential (E) and is also equal to the net charge (Z) of the protein divided by its fractional coefficient (f), which is related to shape.
How does SDS–PAGE ensure that proteins are separated primarily by molecular weight?
SDS coats each protein with a uniform negative charge (approximately one SDS molecule per amino acid), denatures them into rodlike shapes, and masks intrinsic charge, so migration through the gel depends mostly on molecular weight rather than shape or native charge.
What is the typical binding ratio of SDS to protein, and how does that influence electrophoresis?
Proteins bind SDS at about 1.4 times their weight (nearly one SDS molecule per amino acid residue), which imparts a uniform negative charge that makes intrinsic protein charge insignificant.
How are proteins visualized after running an SDS–PAGE gel?
Proteins are stained with Coomassie Brilliant Blue (or non-toxic fluorescent alternatives), which binds to proteins but not the gel matrix, revealing bands corresponding to protein sizes.
Why is SDS necessary when the goal is to measure protein size accurately?
SDS unfolds proteins into rodlike shapes and equalizes their charge-to-mass ratios, eliminating shape and native-charge effects so migration distance reflects molecular weight.
What information does the isoelectric point (pI) of a protein convey?
The pI is the pH at which a protein carries no net charge; below the pI, it is positively charged, and above the pI, it is negatively charged, influencing solubility and behavior in different pH environments.
How can the pI of a protein be determined graphically?
By plotting net protein charge versus pH and identifying the point where the curve crosses zero net charge; that pH is the protein’s pI.
What is isoelectric focusing (IEF) and how does it separate proteins?
IEF establishes a stable pH gradient across a gel using low–molecular-weight acids and bases; proteins migrate until they reach the region where the buffer pH equals their pI, at which point they stop moving.
Why does a protein stop migrating at its pI during IEF?
Because at the pI, the protein has no net charge and thus no electrophoretic mobility, causing it to focus at that position in the pH gradient.
What advantage does combining IEF with SDS–PAGE (2D-PAGE) provide?
2D-PAGE first separates proteins by pI (IEF) and then by molecular weight (SDS–PAGE), allowing resolution of proteins with identical molecular weights but different pI values—or vice versa—with high sensitivity.
What is chromatofocusing, and how does it differ from typical ion-exchange chromatography?
Chromatofocusing is a form of gradient elution that separates proteins based on their pI using ion-exchange resins and a slowly developing pH gradient—unlike typical ion exchange, which elutes proteins by increasing ionic strength.
How is the pH gradient generated during chromatofocusing?
The column is equilibrated with a buffer at a pH above the highest required; then an elution buffer of lower pH is passed through, titrating amines on the resin and attached proteins, creating a continuous pH gradient in situ.
In chromatofocusing, which proteins elute first: those with higher or lower pI?
Proteins with higher pI elute first because, at the starting (higher) pH, they already have net positive charge (pH > pI is negative), so they do not bind strongly and move down the column more quickly.
What is a potential drawback of chromatofocusing when proteins are at high concentration?
Some proteins may aggregate at high concentration, losing their net surface charge; such aggregates do not bind properly and can block the column, preventing proper separation.
Why is chromatofocusing considered useful for “polishing” protein preparations?
Because it can resolve proteins that differ by as little as 0.05 pH units in their pI, enabling separation of very closely related species that remain after bulk purification.
How is column equilibration performed before applying a protein sample in chromatofocusing?
The chromatofocusing medium is flushed with a start buffer at a pH slightly above the highest pH needed, ensuring the column environment is uniform before sample application.
During chromatofocusing, what happens to proteins with pH > pI at the column top?
They are negatively charged at pH above their pI, causing them to be retained near the top of the column until the buffer pH drops to their pI.
What is meant by “gradient elution” in the context of chromatofocusing?
Gradient elution refers to the gradual change in pH of the elution buffer as it moves through the column, which progressively desorbs proteins based on their pI.
What is the void volume (V₀) in size-exclusion chromatography (SEC)?
The void volume is the volume of mobile phase in the column outside the pores; molecules larger than the exclusion limit cannot enter any pores and elute at V₀.
How does SEC separate proteins, and what determines elution order?
SEC separates proteins by size: large proteins bypass resin pores and elute earlier (at or near V₀), whereas smaller proteins enter pores and elute later, with retention volume inversely related to size.
What is the exclusion limit of a size-exclusion resin?
The exclusion limit is the minimum molecular weight above which molecules cannot enter any pores and elute immediately at the void volume.
What is the inclusion limit in SEC?
The inclusion limit is the maximum molecular weight that can enter the entire pore network—molecules smaller than this can access all pores and elute at the total column volume (Vₜ).
How can one estimate a protein’s molecular weight using SEC?
By running molecular weight standards to generate a calibration curve (log MW vs. elution volume), then determining the unknown protein’s elution volume and reading its MW from the curve.
