Exam Prep Electrophoresis Granger Chapter 17

Chapter 17

Problem 17.1: Calculate the force exerted on a singly charged protein in an electro­phoresis experiment if the matrix used is 15 cm long and the applied voltage across the matrix is 20 V.

Problem 17.2: Water will undergo electrolysis at approximately –1.5 V according to the following half reactions:

Suppose you are performing an electrophoresis experiment using an aqueous buffer solution across a 30 cm matrix. What is the maximum voltage you should apply if you want to avoid electrolysis of the solvent buffer?

Problem 17.3: Using a similar approach as demonstrated in Example 17.2, show for two particles, both having the same charge, what the effect on velocity is if one particle is larger, having twice the frictional coefficient of the smaller particle.

Problem 17.4: Electrophoresis experiments conducted in solution—without the use of a matrix—are known as moving boundary or free boundary electrophoresis. Explain what problems would be expected with this experimental design and propose a few reasons why this technique is rarely used today.

Problem 17.5: Finish calculating the electrophoretic mobilities, _ep, for bands B–E in Example 17.3 and create a table of results. (See Example 17.3 on p. 599.)

Problem 17.6: Describe the composition of the following PA gels.

Problem 17.7: Use Table 17.1 to determine which of the gels in Problem 17.6 would be best suited for the separation of relatively small particles. Which would be best suited for the separation of relatively large particles?

Problem 17.8: What would be the correct way to label a gel that is 10% polymer by mass that has 2% cross-linker?

Problem 17.9: If samples are loaded at the negative end of a gel, what happens to positively charged particles in a vertical PAGE experiment?

Problem 17.10: Using the graph in Figure 17.10, determine the molecular mass of a protein fragment that has a relative mobility of 0.75.

Problem 17.11: Compare and contrast the physical parameters of the matrix used in PAGE and agarose electrophoresis. How does the analyst control those physical parameters?

Problem 17.12: Compare and contrast the common detection methods used in liquid and gas chromatography with those used in paper and gel electrophoresis.

Problem 17.13: For the lane labeled “Marker” in Figure 17.14, determine the resolu­tion between the two bands labeled:

(a) LD₂ and LD₃

(b) LD₃₀ and LD₂₉

(c) LD₂₂ and LD₁₇

(d) LD₂₁ and LD₁₈

(e) LD₂₉ and LD₂₈

Problem 17.14: The electrophoretic mobility μ is similar in principle to the k_r value in liquid chromatography (see Chapter 15). Compare and contrast the techniques of chromatography and electrophoresis in the context of the coefficients μ and k_r.

Problem 17.15: How is the separation mechanism in an isoelectric focusing experi­ment fundamentally different from other electrophoresis techniques?

Problem 17.16: One of the most powerful gel electrophoresis techniques is accom­plished by a two-dimensional technique in which a protein mixture is separated by isoelectric focusing in the first dimension, followed by SDS-PAGE in the orthogonal dimension. Describe the resulting information that this experiment provides.

Problem 17.17: The units for E and μ were given in Equations 17.2 and 17.3, respec­tively. By analyzing the units, show that the product of μ_appE in Equation 17.14 does indeed yield a velocity.

Problem 17.18: You are performing a CE experiment with a field strength of 20 V/m and you are separating two analytes (A and B) with μ_app values of 0.001 and 0.0015, respectively. Assume a base line width of 2 seconds. How long must your column be in order to ensure baseline resolution? (See Equation 15.10 for a review of this concept.) Is this a reasonable length? Defend your answer.

Problem 17.19: For a hydrodynamic injected sample, determine the injection volume given, ΔP = 70 kPa, d = 0.25 mm, t = 2 sec, η = 1.3 × 10^–3 Pa·sec, and L = 3 m. If the analyte species were each 3 mM, how many moles of sample were injected?

Problem 17.20: Read the opening profile on Arne Tiselius (“The Father of Electrophoresis”). Discuss how CE is similar to free boundary electrophoresis. Also, discuss how CE is different than free boundary electrophoresis. Be sure to incorporate aspects of the separation mechanisms and resolution in your discussion.

Problem 17.21: Imagine adding thin-layer chromatography to the Compare and Contrast feature “A Look Back at Five Different Separation Techniques” seen directly above. Develop text for the three column headings.

Exercise 17.1: In the gel electrophoresis of a protein sample:

(a) Explain why some proteins migrate farther in a given amount of time than do other proteins?

(b) How does pH affect the migration rates of proteins in a gel electrophoresis experiment?

Exercise 17.2: Sketch an agarose gel for use in separating DNA. Make sure you label:

(a) The location of the wells

(b) The positive electrode

(c) The negative electrode

(d) The direction of migration of the DNA in the gel

Exercise 17.3: Toward which pole would a negatively charged protein migrate (anode or cathode)?

Exercise 17.4: Relative to a protein’s isoelectric point, how would one adjust the pH in order to make the protein migrate toward the cathode?

Exercise 17.5: Relative to a protein’s isoelectric point, how would one adjust the pH in order to make the protein migrate toward the anode?

Exercise 17.6: Why is control of the pH a more impor­tant variable in protein electrophoresis than it is in DNA electrophoresis?

Exercise 17.7: Describe the composition of the follow­ing polyacrylamide gels:

Exercise 17.8: The structures of two common tracking dyes are given below. Under identical conditions, which dye will migrate farther in a gel electropherogram? Defend your answer.

Exercise 17.9: The chemical structure of basic yellow dye #2 is given here. The pK_a1 = 9.8 and the pK_a2 = 10.7. Describe the relative migration rates for an electrophoresis experiment of yellow dye #2 conducted at pH = 8, pH = 10, and pH = 12. Explain.

Exercise 17.10: Draw the structure of ethidium bromide.

Exercise 17.11: What is the main function of ethidium bromide in a gel electrophoresis experiment?

Exercise 17.12: Research and describe three common visualization techniques used in electrophoresis. Cite your references.

Exercise 17.13: What are the theoretical reasons CE enjoys such a high separation efficiency compared to either high-performance liquid chromatography or gas chroma­tography techniques?

Exercise 17.14: Recommend an appropriate gel formu­lation (if appropriate) and an appropriate staining reagent for each of the following analyses:

(a) A paper electrophoresis analysis of proteins with a molecular weight range of 15,000 to 30,000

(b) A Western blot gel electrophoresis analysis of proteins with a molecular weight range of 15,000 to 30,000

(c) Proteins with a molecular weight range of 100,000 to 130,000

(d) A mixture of glycine, leucine, alanine, and proline

(e) Electrophoretic analysis of a mixture of antibody proteins

(f) Genomic analysis of fruit fly DNA subjected to restriction enzymatic digestion

Exercise 17.15: List and describe the three common methods of sample injection used in CE.

Exercise 17.16: Compare and contrast the typical injec­tion volumes needed in slab versus CE.

Exercise 17.17: Sometimes one will observe curved bands in a gel electrophoresis experiment. What experi­mental parameter is responsible for the curvature of the bands?

Exercise 17.18: A composite gel is made of both PA and agarose. How do you think the combination of these mate­rials would affect pore size? Mechanical stability? Melting point? Do a literature search to find an application of a com­posite gel. Does the application meet your expectations?