2. SDS-PAGE & Protein Analysis

What is the main purpose of SDS in SDS-PAGE?

A. To stain proteins blue

B. To denature proteins and give them a broadly uniform negative charge

C. To crosslink acrylamide

D. To reduce the gel pore size

B. To denature proteins and give them a broadly uniform negative charge

Explanation: SDS unfolds proteins and coats them with negative charge so separation is primarily by size.

In SDS-PAGE, proteins migrate toward which electrode?

A. Cathode (negative)

B. Whichever is closer

C. Neither electrode

D. Anode (positive)

D. Anode (positive)

Explanation: SDS-coated proteins are negatively charged and move toward the positive anode.

Which proteins migrate faster through a polyacrylamide gel?

A. Larger proteins

B. More acidic proteins only

C. Smaller proteins

D. More basic proteins only

C. Smaller proteins

Explanation: Smaller proteins pass more readily through the gel mesh.

What is the role of the resolving gel?

A. To separate proteins by molecular weight

B. To concentrate samples into a narrow band

C. To stain proteins

D. To remove SDS

A. To separate proteins by molecular weight

Explanation: The resolving gel provides the size-based separation.

What is the role of the stacking gel?

A. Measure absorbance

B. Determine the protein's pI

C. Digest proteins

D. Normalize/concentrate protein bands before they enter the resolving gel

D. Normalize/concentrate protein bands before they enter the resolving gel

Explanation: The stacking gel focuses samples into narrow bands for improved resolution.

Why is the stacking gel formulated differently from the resolving gel?

A. It uses a different pH and lower acrylamide percentage

B. It contains no water

C. It contains no Tris

D. It is made from agarose

A. It uses a different pH and lower acrylamide percentage

Explanation: The manual describes a ~10% stacking gel with Tris pH 6.8 and a ~15% resolving gel with Tris pH 8.8.

What does DTT or β-mercaptoethanol do in sample preparation?

A. Adds negative charge

B. Breaks disulfide bonds by reduction

C. Stains protein bands

D. Increases sample density

B. Breaks disulfide bonds by reduction

Explanation:7. B — These reducing agents cleave disulfide bonds between cysteine residues.

Why are samples heated to about 95 °C before loading?

A. To polymerize the gel

B. To activate Coomassie

C. To disrupt noncovalent interactions and promote denaturation

D. To remove all amino acids

C. To disrupt noncovalent interactions and promote denaturation

Explanation: Heating helps disrupt hydrogen-bonding and other noncovalent interactions.

What is the purpose of bromophenol blue in the loading buffer?

A. Protein reducing agent

B. Gel crosslinker

C. Protein standard

D. Tracking dye used to monitor the dye front

D. Tracking dye used to monitor the dye front

Explanation:9. D — The dye front helps monitor migration and avoid running proteins off the gel.

Why is glycerol included in the sample buffer?

A. It increases sample density so it settles into the well

B. It reduces disulfide bonds

C. It creates the electric field

D. It stains the gel

A. It increases sample density so it settles into the well

Explanation: Glycerol makes the sample denser than the running buffer.

What do ammonium persulfate (APS) and TEMED contribute to gel casting?

A. Protein staining

B. Protein reduction

C. Initiation/acceleration of acrylamide polymerization and crosslinking

D. Buffering at pH 7.5

C. Initiation/acceleration of acrylamide polymerization and crosslinking

Explanation: APS generates radicals and TEMED promotes polymerization/crosslinking.

Why is the acrylamide solution degassed before polymerization?

A. To remove dissolved oxygen that can interfere with polymerization

B. To add more SDS

C. To increase protein concentration

D. To stain the gel

A. To remove dissolved oxygen that can interfere with polymerization

Explanation: Oxygen inhibits free-radical polymerization, so degassing supports gel formation.

What is the purpose of the protein ladder/standards lane?

A. To measure enzyme activity

B. To increase sample pH

C. To act as the running buffer

D. To provide known molecular-weight bands for comparison

D. To provide known molecular-weight bands for comparison

Explanation: Unknown protein bands are compared with standards to estimate molecular weight.

If a sample band travels farther down an SDS-PAGE gel than a standard band, it is generally:

A. Smaller

B. Larger

C. More concentrated by definition

D. Positively charged

A. Smaller

Explanation: Smaller proteins migrate farther in the same run.

What is the purpose of Coomassie staining after electrophoresis?

A. Make the gel conductive

B. Denature proteins

C. Visualize protein bands

D. Determine the buffer pH

C. Visualize protein bands

Explanation:15. C — Coomassie binds proteins and makes bands visible.

Why is the gel destained after Coomassie staining?

A. To increase protein size

B. To remove the protein bands

C. To reverse electrophoresis

D. To remove excess background stain and improve band contrast

D. To remove excess background stain and improve band contrast

Explanation:16. D — Destaining reduces background dye so protein bands are easier to interpret.

What is the major difference between SDS-PAGE and Native-PAGE?

A. Native-PAGE preserves more native structure/charge differences; SDS-PAGE denatures and standardizes charge

B. Native-PAGE uses no electric field

C. SDS-PAGE separates only DNA

D. They are identical

A. Native-PAGE preserves more native structure/charge differences; SDS-PAGE denatures and standardizes charge

Explanation: SDS-PAGE is denaturing and mainly size-based; Native-PAGE can preserve complexes and native properties.

A protein appears as multiple bands under reducing SDS-PAGE. One possible explanation is:

A. The electric field was absent

B. It contains subunits linked by disulfide bonds or multiple polypeptide species

C. SDS made every protein the same size

D. The ladder was unnecessary

B. It contains subunits linked by disulfide bonds or multiple polypeptide species

Explanation: Reducing conditions can separate disulfide-linked subunits; multiple species or degradation can also produce multiple bands.

Why compare a mystery protein's gel-filtration estimate with its SDS-PAGE estimate?

A. To determine its DNA sequence

B. To calculate its absorbance maximum
C. To assess consistency between native-size behavior and denatured polypeptide size

D. To measure column flow rate

C. To assess consistency between native-size behavior and denatured polypeptide size

Explanation: The techniques rely on different principles and can provide complementary molecular-weight evidence.

In the described SDS-PAGE protocol, why are samples kept on ice before preparation?

A. To help preserve protein samples before denaturation/loading

B. To polymerize acrylamide

C. To increase the gel pore size

D. To make SDS positively charged

A. To help preserve protein samples before denaturation/loading

Explanation: The protocol directs that protein samples be kept cold during preparation.