MCAT Chemical and Physical Foundations of Biological Systems Flashcards: 5c Electrophoresis Protein Separation

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This deck focuses on 5c Electrophoresis Protein Separation, giving you a quick way to review the definitions, rules, and examples that matter most for MCAT Chemical and Physical Foundations of Biological Systems.

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Flashcard 1: If $E$ doubles while gel and sample are unchanged, what happens to velocity $v$ if $\mu$ is constant?

Answer: $v$ doubles because $v = \mu E$. Velocity is directly proportional to electric field at constant mobility.

Flashcard 2: Which gel electrophoresis method separates proteins primarily by isoelectric point ($pI$)?

Answer: Isoelectric focusing (IEF). IEF establishes a pH gradient where proteins migrate until reaching their neutral charge point.

Flashcard 3: What is the main role of $\beta$-mercaptoethanol or DTT in SDS-PAGE?

Answer: Reduces disulfide bonds to separate disulfide-linked subunits. Reducing agents cleave disulfide bonds, allowing individual polypeptide chains to separate.

Flashcard 4: What is the main role of SDS in SDS-PAGE sample preparation?

Answer: Denatures proteins and imparts a uniform negative charge per mass. As an anionic detergent, SDS unfolds proteins and binds proportionally to length, providing consistent negative charge.

Flashcard 5: In SDS-PAGE, what protein property primarily determines migration distance?

Answer: Molecular mass (size), approximately independent of native charge. SDS treatment equalizes charge-to-mass ratios, enabling size-based separation via gel sieving.

Flashcard 6: What is the primary property that determines migration direction in electrophoresis?

Answer: Net electrical charge determines direction toward the opposite electrode. Particles with positive charge migrate to the cathode, while those with negative charge move to the anode.

Flashcard 7: What is electrophoretic mobility ($\mu$) defined as in terms of velocity and field?

Answer: $\mu = \frac{v}{E}$. Mobility quantifies how fast a particle moves per unit electric field strength.

Flashcard 8: What is the relationship between electric field and voltage across a gel of length $L$?

Answer: $E = \frac{V}{L}$. Electric field strength is the voltage gradient over the separation distance.

Flashcard 9: In agarose gel electrophoresis of DNA, which direction do fragments migrate?

Answer: Toward the anode (positive electrode). DNA's phosphate backbone confers a negative charge, attracting fragments to the positive electrode.

Flashcard 10: Identify the method used to estimate protein molecular mass from an SDS-PAGE lane.

Answer: Compare band migration to a molecular weight ladder (standards). Known standards create a reference for interpolating masses based on relative migration distances.

Flashcard 11: What does a Northern blot specifically detect after electrophoretic separation?

Answer: A specific RNA transcript using a nucleic acid probe. After RNA separation and blotting, probes bind specific transcripts for identification.

Flashcard 12: What does a Southern blot specifically detect after electrophoretic separation?

Answer: A specific DNA sequence using a nucleic acid probe. Following DNA gel separation and transfer, labeled probes hybridize to complementary sequences for detection.

Flashcard 13: What does a Western blot specifically detect after electrophoretic separation?

Answer: A specific protein using antibody-based detection. After gel transfer to a membrane, specific antibodies enable visualization of target proteins.

Flashcard 14: In IEF, if a protein is at a region where $\text{pH} > pI$, which direction will it migrate?

Answer: Toward the anode until it reaches the region where $\text{pH} = pI$. Negatively charged above pI, the protein migrates toward the positive electrode in the pH gradient.

Flashcard 15: In IEF, if a protein is at a region where $\text{pH} < pI$, which direction will it migrate?

Answer: Toward the cathode until it reaches the region where $\text{pH} = pI$. Positively charged below pI, the protein moves toward the negative electrode in the pH gradient.

Flashcard 16: Identify the expected charge of a protein at $\text{pH} > pI$ in an electric field.

Answer: Net negative charge. Above pI, basic residues lose protons, while acidic residues deprotonate, yielding overall negative charge.

Flashcard 17: Identify the expected charge of a protein at $\text{pH} < pI$ in an electric field.

Answer: Net positive charge. Below pI, acidic residues are protonated, leaving basic residues to dominate with positive charge.

Flashcard 18: In SDS-PAGE, which band position corresponds to a larger protein: higher or lower in the gel?

Answer: Higher (closer to the wells) corresponds to larger molecular mass. Larger proteins migrate more slowly due to greater resistance from the gel matrix.

Flashcard 19: In gel electrophoresis, how does increasing gel % typically affect separation of small molecules?

Answer: Higher % gel improves resolution of smaller molecules (smaller pores). Denser gels create smaller pores, enhancing the sieving effect for better small molecule discrimination.

Flashcard 20: What is the key difference between agarose gels and polyacrylamide gels in typical MCAT use?

Answer: Agarose for nucleic acids; polyacrylamide for higher-resolution proteins. Agarose's larger pores suit DNA separation, while polyacrylamide's finer matrix resolves smaller proteins better.

Flashcard 21: In 2D-PAGE, what does the first dimension separate by, and what does the second separate by?

Answer: First by $pI$ (IEF), second by molecular mass (SDS-PAGE). IEF separates by charge differences in a pH gradient, followed by size separation under denaturing conditions.

Flashcard 22: Which technique combines IEF and SDS-PAGE to separate proteins in two dimensions?

Answer: Two-dimensional gel electrophoresis (2D-PAGE). This method provides enhanced resolution by using two orthogonal separation criteria.

Flashcard 23: In native PAGE, what protein properties affect migration (choose the best single description)?

Answer: Size, shape, and native net charge all affect migration. Without denaturation, electrophoretic mobility depends on the combined effects of these intrinsic properties.

Flashcard 24: In isoelectric focusing, what happens to a protein when it reaches the pH equal to its $pI$?

Answer: It stops migrating because net charge becomes $0$. Zero net charge eliminates the force from the electric field, halting movement.

Flashcard 25: What is the definition of the isoelectric point ($pI$) of a protein?

Answer: The pH at which the protein has net charge $0$. At this pH, positive and negative charges on the protein balance out.