Announcements Quiz Pre‐lab Lecture More about ELISAAnnouncements • Evaluations – WAC partly in class, end of Lec 7 – Overall subject evaluation available online • Mod 3 report – No separate methods section needed – State anything unique along the way in results section (alginate type, RNA recovered, etc.) • Cross‐group discussion today or next time3ELISA protocol• Direct ELISA uses labeled primary antibody• Indirect ELISA – why use a secondary antibody?• Development process – what/why/how• signal amplification• flexibility (use 2° with many different 1°, efficient)but cross-reactivity more likely2° Ab has enzyme = APprovide substrate = pNPPamplification Æ development time is keycolorimetric reactionELISA Outcomes Outcome Possible Explanations High reading in “blank” samples cross‐contamination * incomplete washes forgot to block No signal at all (including standards) poor systemic binding; inhibitor old reagents too high [Tween]; wrong 1° or 2‐Ab Saturated signal for some samples too concentrated therefore repeat with dilutionsMIT OpenCourseWarehttp://ocw.mit.edu 20.109 Laboratory Fundamentals in Biological Engineering Spring 2010 For information about citing these materials or our Terms of Use, visit:
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