Team:Yale/Project/Crystallography
From 2011.igem.org
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<li>Currently, we are running pH vs. precipitating agent concentration grids, as well as buffering agent vs. precipitating agent concentration grids to optimize crystal growth. In addition, to solve the crystallographic phasing problem, we are using a quick-change, site-directed mutagenesis protocol to replace methionine residues with Se-methione for a reference point. We also plan to crystallize RiAFPs generated through MAGE to determine what structural effects are related to either enhancements or reductions in antifreeze protein activity, e.g. whether smaller/larger helix sheets might allow tighter protein-ice crystal binding to allow improved activity.</li> | <li>Currently, we are running pH vs. precipitating agent concentration grids, as well as buffering agent vs. precipitating agent concentration grids to optimize crystal growth. In addition, to solve the crystallographic phasing problem, we are using a quick-change, site-directed mutagenesis protocol to replace methionine residues with Se-methione for a reference point. We also plan to crystallize RiAFPs generated through MAGE to determine what structural effects are related to either enhancements or reductions in antifreeze protein activity, e.g. whether smaller/larger helix sheets might allow tighter protein-ice crystal binding to allow improved activity.</li> | ||
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+ | <h1>Figures</h1> | ||
+ | <img src="https://static.igem.org/mediawiki/2011/b/b1/Protein1.jpg" style="margin-top:5px; margin-left:auto; margin-right:auto; display:block;" /> | ||
+ | <center>Figure 1: SDS-PAGE stained with Coomassie blue; uninduced eGFP-TmAFP (left), induced eGFP-TmAFP (right)</center> | ||
+ | </div> | ||
+ | <div id="container"> | ||
+ | <img src="https://static.igem.org/mediawiki/2011/5/55/Yale-Protein2.jpg" style="margin-top:5px; margin-left:auto; margin-right:auto; display:block;" /> | ||
+ | <center>Figure 2: Western Blot, probed with His-6 antibody; uninduced eGFP-TmAFP (left), induced eGFP-TmAFP (right)</center> | ||
+ | </div> | ||
+ | <div id="container"> | ||
+ | <img src="https://static.igem.org/mediawiki/2011/8/85/Yale-Protein3.jpg" style="margin-top:5px; margin-left:auto; margin-right:auto; display:block;" /> | ||
+ | <center>Figure 3: Western Blot, probed with eGFP antibody. Lane 0: ladder, Lane 1: uninduced eGFP-RiAFP in BL21, Lane 2: induced eGFP-RiAFP in BL21, Lane 3: uninduced eGFP-RiAFP in Origami, Lane 4: induced eGFP-RiAFP in Origami, Lane 5: uninduced RiAFP in BL21, Lane 6: induced RiAFP in BL21, Lane 7: uninduced RiAFP in Origami, Lane 8: induced RiAFP in Origami, Lane 9: uninduced eGFP-TEV-TmAFP, Lane 10: induced eGFP-TEV-TmAFP, Lane 11: ladder</center> | ||
+ | </div> | ||
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Revision as of 01:10, 29 September 2011