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Lab Notebook and Experiment Summary for Gel Electrophoresis and Ultracentrifugation Techniques

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This week, lab notebooks are due, and students must submit a one-paragraph summary of their denaturation experiment as a pair. Additionally, each pair will sign up for 25 minutes of Computational Design (CD) time on Wednesday and Thursday. The experiment includes utilizing dialysis methods to separate small and large molecules and covers key figures and tables related to gel electrophoresis and ultracentrifugation. Understanding structural features of globular proteins, species separation based on density, and molecular mobility in SDS-PAGE is also crucial for this week's discussions.

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Lab Notebook and Experiment Summary for Gel Electrophoresis and Ultracentrifugation Techniques

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  1. Today: Lab notebooks due Next week: M/T analyze gels Computer lab Turn in a one paragraph summary of your denaturation experiment (per PAIR) W/R CD: each PAIR will sign up for 25 minutes of CD time

  2. Figure 6-11 Use of dialysis to separate small and large molecules. Page 139

  3. Figure 6-29 A selection of preparative ultracentrifuge rotors. Page 154

  4. Figure 6-32 Isopycnic ultracentrifugation. Page 155 components of a mixture are separated on the basis of differences in density

  5. Figure 6-28 Sedimentation coefficients in Svedbergs (S) for some biological materials. Page 153

  6. Table 6-5 Physical Constants of Some Proteins. Page 152

  7. Table 6-4 Purification of Rat Liver Glucokinase. Page 142

  8. Theoretical considerations http://www.bergen.org/AAST/Projects/Gel/technique2.htm

  9. Ammonium persulfate: initiates the polymerization NH4SO3-O-O-SO3NH4 S2O8-2 + 1e- SO4-2 + SO4-• Free radical TEMED: N,N-tetramethylenediamnine: catalyst (CH3)2- N-CH2-CH2-N(CH3)2 • SO4-• + n CH2=CH-CONH2 X-CH2-CH-CONH2 • X-CH2-CH(CONH2)-CH2-CH(CONH2)-CH-CONH2

  10. Figure 6-20 Apparatus for slab gel electrophoresis. Page 146

  11. CH3-(CH2)11-SO4- Na+

  12. Structural features of most globular proteins: 1. Very compact: e.g. Mb has room for only4 water molecules in its interior. 2. Most polar/charged R groups are on the surface and are hydrated. 3. Nearly all the hydrophobic R groups are on the interior. 4. Pro occurs at bends/loops/random structures and in sheets

  13. Simulation http://www.rit.edu/~pac8612/electro/Electro_Sim.html

  14. Figure 6-25 Logarithmic relationship between the molecular mass of a protein and its relative electrophoretic mobility in SDS-PAGE. log

  15. Proteins in native gels exhibit a more complex relationship between mobility and MW.

  16. Figure 6-27 Two-dimensional (2D) gel electrophoresis. Page 150

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