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Reminder about lab notebooks due today. Next week, analyze gels in the computer lab. Write a summary of your denaturation experiment for each pair. Sign up for CD time. Learn about ultracentrifugation, dialysis, and gel electrophoresis components. Explore protein purification techniques and simulation. Understand protein structure features and electrophoretic mobility. For more details, visit the provided links.
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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
Figure 6-11 Use of dialysis to separate small and large molecules. Page 139
Figure 6-29 A selection of preparative ultracentrifuge rotors. Page 154
Figure 6-32 Isopycnic ultracentrifugation. Page 155 components of a mixture are separated on the basis of differences in density
Figure 6-28 Sedimentation coefficients in Svedbergs (S) for some biological materials. Page 153
Theoretical considerations http://www.bergen.org/AAST/Projects/Gel/technique2.htm
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
Figure 6-20 Apparatus for slab gel electrophoresis. Page 146
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
Simulation http://www.rit.edu/~pac8612/electro/Electro_Sim.html
Figure 6-25 Logarithmic relationship between the molecular mass of a protein and its relative electrophoretic mobility in SDS-PAGE. log
Proteins in native gels exhibit a more complex relationship between mobility and MW.
Figure 6-27 Two-dimensional (2D) gel electrophoresis. Page 150