Q: Principle of SDS–PAGE — On what fundamental property does SDS–PAGE separate protein species?

Introduction: SDS–PAGE is designed to compare polypeptides primarily by size. SDS binds and unfolds proteins, minimizing effects of native charge and shape so that separation reflects length (mass) of the polypeptide chain. Given Data / Assumptions: SDS confers a near-constant negative charge-to-mass ratio. Reducing agents may be included to break disulfide bonds. Electrophoretic mobility in the gel is inversely related to log(molecular mass). Concept / Approach: Because charge is standardized by SDS binding, migration differences are predominantly due to size. Smaller proteins travel farther through the polyacrylamide matrix within a fixed time frame. Step-by-Step Solution: 1) SDS coats polypeptides uniformly → standardizes charge.2) Denaturation removes tertiary/quaternary structure influences.3) Migration distance correlates with molecular mass; bands can be compared to standards to estimate mass. Verification / Alternative check: Plotting migration distance against log(MW) for standards produces a calibration curve used to estimate unknowns. Why Other Options Are Wrong: a,c) Individual side-chain charges are masked by SDS.d) pI differences are resolved by isoelectric focusing, not SDS–PAGE.e) Native structures are disrupted; SDS–PAGE is not a native method. Common Pitfalls: Assuming glycosylation or extreme hydrophobicity never affect mobility; unusual modifications can cause anomalies, but the core principle remains mass-based separation. Final Answer: Molecular weight (apparent mass).

Q: SDS–PAGE characteristics — Identify the set of statements that correctly describe protein behavior in SDS–PAGE

Introduction: This question checks recognition of three hallmark features of SDS–PAGE: denaturation, charge normalization, and size-dependent mobility. Understanding these together explains why SDS–PAGE estimates apparent molecular masses reliably. Given Data / Assumptions: SDS is an anionic detergent that unfolds proteins. SDS binding is roughly proportional to length, producing near-constant charge-to-mass ratios. Polyacrylamide gels sieve molecules by size under an electric field. Concept / Approach: Combine the chemical action of SDS with the physical sieving of the gel. Denatured chains with similar charge density migrate according to length, so smaller chains move faster and farther during a set run time. Step-by-Step Solution: 1) Confirm denaturation effect (true) → supports removal of shape effects.2) Confirm constant charge density (true) → eliminates native charge differences.3) Confirm size dependence (true) → explains separation principle. Verification / Alternative check: Standard marker ladders yield linear relationships between distance migrated and log(MW), empirically validating the trio of statements. Why Other Options Are Wrong: e) SDS–PAGE does not preserve native assemblies; that is a property of native PAGE or BN-PAGE. Common Pitfalls: Assuming reducing agents are always required; some analyses run non-reducing SDS–PAGE when disulfide status is of interest. Final Answer: All of the above.

Q: Determining subunit stoichiometry — Which combination of methods lets you find both subunit molecular weight and the number of subunits in a protein's quaternary structure?

Introduction: Determining quaternary structure often requires knowing both the mass of the intact native assembly and the masses of its individual subunits. Integrating orthogonal separation data provides a practical route to subunit stoichiometry. Given Data / Assumptions: SDS–PAGE estimates individual polypeptide molecular weights under denaturing conditions. Size-exclusion (gel filtration) estimates the native molecular weight of the intact complex. Subunit count ≈ native MW / dominant subunit MW (accounting for heterogeneity if present). Concept / Approach: Use SDS–PAGE to identify subunit sizes and relative band intensities; use gel filtration to estimate the native complex mass. The ratio provides an integer consistent with known subunit composition, validated against standards and known oligomeric forms. Step-by-Step Solution: 1) Run SDS–PAGE → determine subunit masses (e.g., 25 kDa and 30 kDa).2) Run gel filtration → determine native complex (e.g., ~110 kDa).3) Compare: 110 kDa vs subunits implies likely stoichiometries (e.g., 2 * 25 + 2 * 30 = 110 → heterotetramer). Verification / Alternative check: Cross-check with native PAGE or mass spectrometry. Non-reducing SDS–PAGE can reveal disulfide-linked oligomers complementing gel filtration data. Why Other Options Are Wrong: a) Reveals subunit MW but not intact native mass.b) Reveals native mass but not individual subunits.d) Resolves by pI, not mass; insufficient for stoichiometry alone.e) Lacks calibration and denatured subunit information. Common Pitfalls: Interpreting anomalous elution due to shape or glycosylation as true mass; ignoring hetero-oligomer composition when dividing masses. Final Answer: Combine SDS–PAGE with gel filtration.

Q: Isoelectric focusing principle — Proteins are separated primarily based on what compositional/physicochemical property?

Introduction: Isoelectric focusing (IEF) separates proteins along a fixed pH gradient until each focuses at its isoelectric point, the pH where net charge equals zero. Understanding pI connects sequence composition to separations in proteomics workflows. Given Data / Assumptions: A stable pH gradient exists across the gel or capillary. Proteins migrate under an electric field until net charge is zero. pI depends on the balance of acidic and basic residues and their pKa values. Concept / Approach: Because migration stops when net charge is zero, proteins resolve sharply at pI values. This is complementary to SDS–PAGE (size-based) and can be combined in 2D electrophoresis (IEF in the first dimension, SDS–PAGE in the second). Step-by-Step Solution: 1) Apply electric field → proteins move according to instantaneous charge at local pH.2) Each protein reaches the pH where positive and negative charges balance (pI) → net charge 0.3) Focusing occurs because any displacement from pI restores charge and drives the protein back. Verification / Alternative check: Mixing pI markers with samples confirms gradient integrity and focusing performance; spots align with expected pI values in 2D maps. Why Other Options Are Wrong: a,b) pI reflects both positive and negative residues, not one class alone.c) Size is not the IEF basis; that is SDS–PAGE.e) Hydrophobicity affects chromatography with detergents, not IEF. Common Pitfalls: Assuming mobility ceases for all proteins at the same pH; overlooking ampholyte gradient stability; confusing IEF with charge-based native PAGE lacking a fixed gradient. Final Answer: Separation is by isoelectric point (pI), reflecting the overall balance of positive and negative residues.

Q: Size-exclusion (gel filtration) chromatography — Predict elution order and bead access for proteins of different sizes

Introduction: Gel filtration separates macromolecules by hydrodynamic size. The stationary phase contains porous beads; accessibility to pore volume determines retention and elution order. Given Data / Assumptions: Beads have defined fractionation ranges. Large proteins are excluded from more pores than small proteins. Mobile phase carries solutes through the packed bed. Concept / Approach: Exclusion from pores reduces path length, so very large proteins traverse mainly the interstitial volume and elute early. Smaller proteins take longer paths by entering pores, eluting later. This principle enables estimation of native molecular weight using standards. Step-by-Step Solution: 1) Consider pore accessibility: small > medium > large.2) Translate to elution volume: large proteins elute at lower volumes (earlier), small proteins at higher volumes (later).3) Therefore, statements 'smaller enter beads more readily' and 'large elute first' are both true. Verification / Alternative check: Plotting elution volume vs log(MW) with standards yields a calibration curve used to infer native size for unknowns. Why Other Options Are Wrong: d) Opposite of the size-exclusion mechanism.e) Proteins do enter beads if their size is within the fractionation range. Common Pitfalls: Ignoring shape effects (elongated proteins behave larger); operating outside the bead fractionation range; interpreting nonspecific interactions as size effects. Final Answer: Both (a) and (b).

Question 1

Electrophoresis basics — In native PAGE, proteins are separated primarily on the basis of what properties?

Accepted Answer

Introduction: Electrophoresis separates biomolecules by their movement in an electric field through a gel matrix. This question focuses on native PAGE, where proteins retain their folded state and intrinsic charges, in contrast to denaturing SDS PAGE. Given Data / Assumptions: Native PAGE preserves protein conformation and complexes. No uniform charge to mass ratio is imposed by detergents. Gel matrix introduces size dependent friction. Concept / Approach: In native PAGE, mobility reflects a balance between electrical force and frictional drag: v ∝ (q * E) / f, where q is net charge and f depends on size and shape. Larger complexes experience greater friction and move more slowly; proteins with greater net charge in the running buffer move faster toward the opposite electrode. Step-by-Step Solution: 1) Identify forces: the electric field accelerates proteins according to their net charge at the buffer pH.2) Recognize resistance: the gel matrix introduces size and shape dependent friction.3) Conclude: separation depends on both net charge and hydrodynamic size (and shape), not exclusively on charge sign.4) Therefore, the best description is net charge and size. Verification / Alternative check: Comparing a protein and a larger complex with the same charge shows slower migration of the larger species. Changing buffer pH alters net charge and shifts mobility, confirming the role of q and size together. Why Other Options Are Wrong: Net negative charge or net positive charge alone: sign by itself does not define mobility or separation without magnitude and size. Net positive charge and size: restricts sign unnecessarily; proteins can be negative or positive depending on pH. Isoelectric point only: relevant for isoelectric focusing, not native PAGE in a constant pH gradient. Common Pitfalls: Confusing native PAGE with SDS PAGE. In SDS PAGE, size dominates due to near constant charge to mass ratio; in native PAGE, both charge and size (and shape) matter significantly. Final Answer: net charge and size.

Question 2

SDS–PAGE workflow in protein biochemistry — What is the correct initial sample treatment before electrophoretic separation on a polyacrylamide gel containing the anionic detergent SDS?

Accepted Answer

Introduction: SDS–PAGE is a foundational technique to separate proteins primarily by molecular mass. The preparative chemistry before loading is critical: proteins are denatured and, if required, disulfide bonds are reduced to ensure subunits migrate according to size rather than shape or native charge. Given Data / Assumptions: SDS is an anionic detergent that binds proteins roughly in proportion to length. Reducing agents such as β-mercaptoethanol or DTT cleave disulfide bonds. Electrophoresis is performed in a polyacrylamide matrix under an electric field. Concept / Approach: SDS unfolds proteins and confers a near-constant negative charge-to-mass ratio. Reducing agents remove disulfide constraints so each polypeptide chain migrates independently. Correct sample preparation precedes electrophoresis to standardize migration behavior across diverse proteins. Step-by-Step Solution: 1) Mix sample with SDS-containing sample buffer to denature and coat polypeptides.2) Add a reducing agent (β-mercaptoethanol or DTT) to break disulfide bonds; heating often assists denaturation.3) Load the treated sample into wells and apply voltage to separate mainly by molecular mass. Verification / Alternative check: When reduction is omitted, covalently linked subunits co-migrate as larger species. Including reducing agent yields band patterns that match subunit molecular masses expected from known sequences. Why Other Options Are Wrong: b) Treatment after electrophoresis is illogical; separation must follow denaturation.c) Oxidizing agents create or preserve disulfides, counter to SDS–PAGE goals.d) Native conditions do not reflect SDS–PAGE methodology.e) Dialysis and native PAGE would separate by charge/shape, not SDS mass-based separation. Common Pitfalls: Confusing native PAGE with SDS–PAGE; neglecting reduction when analyzing multimeric proteins; underheating samples leading to incomplete denaturation. Final Answer: Treat with a reducing agent and SDS, then perform electrophoresis.

Question 3

Native (non-denaturing) electrophoresis at pH 7.0 — Predict the migration directions of histones and myoglobin based on net charge

Accepted Answer

Introduction: In native electrophoresis, proteins migrate according to their net charge and size at the working pH. This question tests understanding of isoelectric points and how pH relative to pI determines the direction of migration in an electric field. Given Data / Assumptions: pH is 7.0 (near physiological). Histones are rich in Lys and Arg, giving high pI values (typically > 10). Myoglobin has a pI near neutral but is slightly below or around 7.0 depending on species; at pH 7.0 it is near neutral to slightly negative. Anode is the positive electrode; cathode is the negative electrode. Concept / Approach: Rule of thumb: if pH > pI, the protein carries net negative charge and migrates toward the anode. If pH < pI, the protein carries net positive charge and migrates toward the cathode. Compare pH 7.0 to each protein’s pI to determine direction. Step-by-Step Solution: 1) Histones: pI is far above 7.0, so pH < pI → net positive → migrate toward the cathode.2) Myoglobin: pI is approximately around 6.8–7.2 in many preparations. At pH 7.0, it is near neutral; slight negativity is typical in many buffers → migrate toward the anode (albeit slowly).3) Therefore, histones to the cathode and myoglobin to the anode is the best prediction. Verification / Alternative check: Running standards under native conditions commonly shows basic proteins moving toward the cathode, while acidic or slightly acidic proteins move to the anode. Minimal mobility occurs when pH equals pI exactly. Why Other Options Are Wrong: a) Both to the anode ignores histones’ high basicity.b) Reverses charges for both proteins.d) Both to the cathode ignores myoglobin’s near-neutral to slightly acidic nature at pH 7.e) Zero migration would require exact pH = pI for both, which is not the case for histones. Common Pitfalls: Confusing electrode polarity; assuming myoglobin is neutral and immobile; overlooking that even small deviations from pI impart directionality, especially over longer runs. Final Answer: Histones migrate to the cathode; myoglobin migrates to the anode.

Question 4

Principle of SDS–PAGE — On what fundamental property does SDS–PAGE separate protein species?

Accepted Answer

Introduction: SDS–PAGE is designed to compare polypeptides primarily by size. SDS binds and unfolds proteins, minimizing effects of native charge and shape so that separation reflects length (mass) of the polypeptide chain. Given Data / Assumptions: SDS confers a near-constant negative charge-to-mass ratio. Reducing agents may be included to break disulfide bonds. Electrophoretic mobility in the gel is inversely related to log(molecular mass). Concept / Approach: Because charge is standardized by SDS binding, migration differences are predominantly due to size. Smaller proteins travel farther through the polyacrylamide matrix within a fixed time frame. Step-by-Step Solution: 1) SDS coats polypeptides uniformly → standardizes charge.2) Denaturation removes tertiary/quaternary structure influences.3) Migration distance correlates with molecular mass; bands can be compared to standards to estimate mass. Verification / Alternative check: Plotting migration distance against log(MW) for standards produces a calibration curve used to estimate unknowns. Why Other Options Are Wrong: a,c) Individual side-chain charges are masked by SDS.d) pI differences are resolved by isoelectric focusing, not SDS–PAGE.e) Native structures are disrupted; SDS–PAGE is not a native method. Common Pitfalls: Assuming glycosylation or extreme hydrophobicity never affect mobility; unusual modifications can cause anomalies, but the core principle remains mass-based separation. Final Answer: Molecular weight (apparent mass).

Question 5

SDS–PAGE characteristics — Identify the set of statements that correctly describe protein behavior in SDS–PAGE

Accepted Answer

Introduction: This question checks recognition of three hallmark features of SDS–PAGE: denaturation, charge normalization, and size-dependent mobility. Understanding these together explains why SDS–PAGE estimates apparent molecular masses reliably. Given Data / Assumptions: SDS is an anionic detergent that unfolds proteins. SDS binding is roughly proportional to length, producing near-constant charge-to-mass ratios. Polyacrylamide gels sieve molecules by size under an electric field. Concept / Approach: Combine the chemical action of SDS with the physical sieving of the gel. Denatured chains with similar charge density migrate according to length, so smaller chains move faster and farther during a set run time. Step-by-Step Solution: 1) Confirm denaturation effect (true) → supports removal of shape effects.2) Confirm constant charge density (true) → eliminates native charge differences.3) Confirm size dependence (true) → explains separation principle. Verification / Alternative check: Standard marker ladders yield linear relationships between distance migrated and log(MW), empirically validating the trio of statements. Why Other Options Are Wrong: e) SDS–PAGE does not preserve native assemblies; that is a property of native PAGE or BN-PAGE. Common Pitfalls: Assuming reducing agents are always required; some analyses run non-reducing SDS–PAGE when disulfide status is of interest. Final Answer: All of the above.

Question 6

Determining subunit stoichiometry — Which combination of methods lets you find both subunit molecular weight and the number of subunits in a protein's quaternary structure?

Accepted Answer

Introduction: Determining quaternary structure often requires knowing both the mass of the intact native assembly and the masses of its individual subunits. Integrating orthogonal separation data provides a practical route to subunit stoichiometry. Given Data / Assumptions: SDS–PAGE estimates individual polypeptide molecular weights under denaturing conditions. Size-exclusion (gel filtration) estimates the native molecular weight of the intact complex. Subunit count ≈ native MW / dominant subunit MW (accounting for heterogeneity if present). Concept / Approach: Use SDS–PAGE to identify subunit sizes and relative band intensities; use gel filtration to estimate the native complex mass. The ratio provides an integer consistent with known subunit composition, validated against standards and known oligomeric forms. Step-by-Step Solution: 1) Run SDS–PAGE → determine subunit masses (e.g., 25 kDa and 30 kDa).2) Run gel filtration → determine native complex (e.g., ~110 kDa).3) Compare: 110 kDa vs subunits implies likely stoichiometries (e.g., 2 * 25 + 2 * 30 = 110 → heterotetramer). Verification / Alternative check: Cross-check with native PAGE or mass spectrometry. Non-reducing SDS–PAGE can reveal disulfide-linked oligomers complementing gel filtration data. Why Other Options Are Wrong: a) Reveals subunit MW but not intact native mass.b) Reveals native mass but not individual subunits.d) Resolves by pI, not mass; insufficient for stoichiometry alone.e) Lacks calibration and denatured subunit information. Common Pitfalls: Interpreting anomalous elution due to shape or glycosylation as true mass; ignoring hetero-oligomer composition when dividing masses. Final Answer: Combine SDS–PAGE with gel filtration.

Question 7

Isoelectric focusing principle — Proteins are separated primarily based on what compositional/physicochemical property?

Accepted Answer

Introduction: Isoelectric focusing (IEF) separates proteins along a fixed pH gradient until each focuses at its isoelectric point, the pH where net charge equals zero. Understanding pI connects sequence composition to separations in proteomics workflows. Given Data / Assumptions: A stable pH gradient exists across the gel or capillary. Proteins migrate under an electric field until net charge is zero. pI depends on the balance of acidic and basic residues and their pKa values. Concept / Approach: Because migration stops when net charge is zero, proteins resolve sharply at pI values. This is complementary to SDS–PAGE (size-based) and can be combined in 2D electrophoresis (IEF in the first dimension, SDS–PAGE in the second). Step-by-Step Solution: 1) Apply electric field → proteins move according to instantaneous charge at local pH.2) Each protein reaches the pH where positive and negative charges balance (pI) → net charge 0.3) Focusing occurs because any displacement from pI restores charge and drives the protein back. Verification / Alternative check: Mixing pI markers with samples confirms gradient integrity and focusing performance; spots align with expected pI values in 2D maps. Why Other Options Are Wrong: a,b) pI reflects both positive and negative residues, not one class alone.c) Size is not the IEF basis; that is SDS–PAGE.e) Hydrophobicity affects chromatography with detergents, not IEF. Common Pitfalls: Assuming mobility ceases for all proteins at the same pH; overlooking ampholyte gradient stability; confusing IEF with charge-based native PAGE lacking a fixed gradient. Final Answer: Separation is by isoelectric point (pI), reflecting the overall balance of positive and negative residues.

Question 8

Size-exclusion (gel filtration) chromatography — Predict elution order and bead access for proteins of different sizes

Accepted Answer

Introduction: Gel filtration separates macromolecules by hydrodynamic size. The stationary phase contains porous beads; accessibility to pore volume determines retention and elution order. Given Data / Assumptions: Beads have defined fractionation ranges. Large proteins are excluded from more pores than small proteins. Mobile phase carries solutes through the packed bed. Concept / Approach: Exclusion from pores reduces path length, so very large proteins traverse mainly the interstitial volume and elute early. Smaller proteins take longer paths by entering pores, eluting later. This principle enables estimation of native molecular weight using standards. Step-by-Step Solution: 1) Consider pore accessibility: small > medium > large.2) Translate to elution volume: large proteins elute at lower volumes (earlier), small proteins at higher volumes (later).3) Therefore, statements 'smaller enter beads more readily' and 'large elute first' are both true. Verification / Alternative check: Plotting elution volume vs log(MW) with standards yields a calibration curve used to infer native size for unknowns. Why Other Options Are Wrong: d) Opposite of the size-exclusion mechanism.e) Proteins do enter beads if their size is within the fractionation range. Common Pitfalls: Ignoring shape effects (elongated proteins behave larger); operating outside the bead fractionation range; interpreting nonspecific interactions as size effects. Final Answer: Both (a) and (b).

Question 9

Visualizing proteins in polyacrylamide gels — Which approach directly reveals protein bands after electrophoresis?

Accepted Answer

Introduction: After electrophoresis, proteins in polyacrylamide gels are typically invisible. Visualization requires dyes or labels that bind proteins to produce contrast against the clear gel background. Given Data / Assumptions: Most proteins lack intrinsic color at low concentrations. Common dyes include Coomassie Brilliant Blue and silver stain; fluorescent stains are also used. Gels must often be destained to reduce background. Concept / Approach: Dyes interact non-covalently with proteins, allowing discrete bands to be seen and documented. Sensitivity and linearity vary by stain, influencing quantitation and detection limits. Step-by-Step Solution: 1) Run electrophoresis to separate proteins by mass (SDS–PAGE) or by other criteria (native/IEF).2) Immerse gel in staining solution to bind dye to protein bands.3) Destain until background clears and bands are visible for imaging and analysis. Verification / Alternative check: Include molecular weight standards; their visible bands confirm staining quality and enable mass estimation by comparison. Why Other Options Are Wrong: b) Electron microscopy is not a standard method for intact, hydrated gels.c) Sequence-based estimates do not generate visual bands.d) Most proteins are colorless at analytical concentrations.e) Electrical measurements do not visualize band positions. Common Pitfalls: Overstaining leading to high background; under-destaining obscuring bands; neglecting fixation steps when required by the staining protocol. Final Answer: Stain the gel with a protein-binding dye to visualize bands.

Gel Electrophoresis Questions