44X+W5+Postlab+Instructions - Bio44X Week 5 Week 5 Post-lab...

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Bio44X Week 5 Week 5 Post-lab Assignment Part 1: Analysis of your Ponceau S-stained Western Blot Membrane Open the document, 44X Postlab 5 Template.doc, in Coursework and save it as a .doc, .docx or .pages file. QUESTION Q1: (5pts) Briefly describe what question you are trying to address by performing a western blot on your p53 mutant. RESULTS Q2: Prepare a figure of your Ponceau S stained membrane (5 pts) A) First, rotate and/or flip the scan of your membrane so that the colored protein standards in lane 1 are shown on the left, with the bands corresponding to the largest proteins at the top and the smallest ones at the bottom (unless you transferred differently than indicated in the protocol, in which case your ladder may be on the right). Then, crop your figure to include only the nitrocellulose membrane. Using a graphics program such as PowerPoint, Photoshop, or Illustrator, add 1) labels indicating the molecular weights of the protein markers (which can be found in 44X W5 Background.pdf) to the left of the membrane, 2) the p53 allele (wt, mut# or ) on top, over the corresponding lanes, and 3) the growth temperature (30 o C or 36 o C) of the yeast from which the extracts were prepared. Save your labeled Ponceau S figure as a .jpg file and insert it into your template file under Q2. YOUR INTERPRETATION Q3: (10 pts) Briefly describe what, if anything, the results of your Ponceau S staining of the membrane tell you about the amount of (1) total protein and (2) p53 in each of your yeast extracts. Q4: (10 pts) Briefly explain why you transformed a p53-mut/sfGFP plasmid into the yeast strain, DH124. Be sure to specifically address the following two questions: 1) What information about your mutant p53 will your p53- mut/sfGFP yeast strain ultimately give you? 2) Recall that your yeast host strain has a Consensus p53- RE/ ADE2 reporter gene. What information about your mutant will this allow you to learn next week ?
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  • Fall '13
  • Talbot
  • ADE2 spot assay

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