Electrophoresis and Protein Separation (5C) - Chem & Physics

1. What is the main role of SDS in SDS-PAGE sample preparation?

   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.

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

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

3. In 2D-PAGE, what does the first dimension separate by, and what does the second separate by?

   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.

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

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

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

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

6. Which technique combines IEF and SDS-PAGE to separate proteins in two dimensions?

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

7. What is the relationship between electric field and voltage across a gel of length $L$?

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

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

   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.

9. In isoelectric focusing, what happens to a protein when it reaches the pH equal to its $pI$?

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

10. What is the primary property that determines migration direction in electrophoresis?

    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.

11. In gel electrophoresis, how does increasing gel % typically affect separation of small molecules?

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

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

    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.

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

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

14. In SDS-PAGE, what protein property primarily determines migration distance?

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

15. In native PAGE, what protein properties affect migration (choose the best single description)?

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

16. What does a Northern blot specifically detect after electrophoretic separation?

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

17. What is the key difference between agarose gels and polyacrylamide gels in typical MCAT use?

    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.

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

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

19. In agarose gel electrophoresis of DNA, which direction do fragments migrate?

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

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

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

21. What is the definition of the isoelectric point ($pI$) of a protein?

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

22. What does a Southern blot specifically detect after electrophoretic separation?

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

23. What is the main role of $\beta$-mercaptoethanol or DTT in SDS-PAGE?

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

24. Which gel electrophoresis method separates proteins primarily by isoelectric point ($pI$)?

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

25. What is electrophoretic mobility ($\mu$) defined as in terms of velocity and field?

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

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