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BME 103 Homework 4 Total Points: 100 Due in class Tues 3-18-2014 1.

(10 Points) Given that a hexane-based liquid has lv of ~30 dynes/cm, which of the two materials depicted below is more hydrophobic? Explain in terms of how the Zisman method is used to determine critical surface tension and what this represents in terms of surface properties.

2. (20 Points) You have a mixture of two proteins that you are trying to separate via affinity chromatography. X contains many serine residues (Polar) Y contains many phenylalanine residues (Nonpolar) a. (5 pts) You are using a reversed phase system with water as the mobile phase. Which protein would you expect to elute first? Explain based on molecular interactions with the column. b. 5 pts) If you switch to using methanol as the mobile phase, will this make it easier or harder to separate the two proteins? Explain based on the table below and molecular interaction with the column.

c. (10 pts) After optimizing your reversedphased HPLC system, you run standard samples of protein X. After running the standards, three samples (A, B and C) of protein X of unknown concentration were run through the system as noted below:

Calculate the concentrations of each of the unknown protein samples. Make all steps explicit in the generation of your answer, including where in the process the Beer Lambert law is used. 3. (20 Points) Of the surface analysis methods discussed in chapter 7, which require physical contact with the material surface? Which do not? Which can identify atomic compositions, and how? Which can identify functional groups, and how? 4. (10 Points) One potential problem associated with the long-term release of a drug from an implanted material is the initial burst release of significant amounts of the drug. One approach to reduce this burst release is to apply a coating to the material in an attempt to modulate the diffusion of the drug. In a study performed by Kwok et al. [24] the effectiveness of butyl methacrylate as a coating material was tested. Figure 1 below shows the chemical structure and a ESCA scan of the surface of this coating material. Plasma treatment of its surface was also performed in an attempt to increase its hydrophilicity. Part of the study explored how the power of the plasma-generating equipment affects the surface chemistry (Fig. 2 below). At high power, some of the polymer begins to degrade. Which of the bonds in the polymer is subject to degradation at high power? Explain in terms of the results in Fig. 2. Which power setting would you recommend for surface treating this polymer and why? FIGURE 1

FIGURE 2

Legend: (a) untreated polymer (b-g) plasma-treated polymer: (b) 5W, (c) 10W, (d) 20W, (e) 40W, (f) 50W, (g) 60W 5. (20 Points) You are performing an in vitro experiment in which you will expose a material you are considering for a medical device to synovial fluid, which contains the proteins albumin, transferrin, and IgM at concentrations of 5, 0.5, and 0.05 mg/ml, respectively. Each of these components has a particular affinity for your material, with IgM being the highest and albumin being the lowest. a. Describe the kinetics of protein adsorption to your material and how the surface concentration of each protein will change with time. Provide a hypothetical diagram of the Vroman effect with each curve labeled with the associated protein. (Hint: See Figure 8.19) b. Subsequently in the experiment, more albumin is added to the synovial fluid. What effect will this have on the proteins adsorbed on the surface? c. The graph below shows the amount of IgM adsorbed to the material surface as a function of time. What will happen on the material surface with the addition of more IgM? 6. (20 Points) Describe the 6 major steps for preforming an ELISA. You have gone out of town for the week and your labmate needed to borrow your reagents for a sandwich ELISA for the antigen p24. Unfortunately, the labmate mixed your reagents with theirs and didnt write down their protocol. Redesign your ELISA, choosing the appropriate reagents from the list below (wont use all the reagents): Goat anti-p24 (epitope 2) mAb

Streptavidin-Horseradish Peroxidase (HRP) Rabbit anti-p24 (epitope 2) mAb Biotinylated p24 Biotinylated rabbit anti-goat Fc Mouse anti-p24 (epitope 1) mAb Rat anti-p24 (epitope 1) mAb Rabbit anti-mouse Fc p24