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Molecular Biology of the Cell

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Unformatted text preview: Define four new science related words found in this paper. What was this paper about? What was this paper about? Describe the problem being addressed. Cystic Fibrosis Transmembrane Conductance Cystic Regulator (CFTR) Regulator Many types of mutation effects gene Delta F508 mutation ? What % of proteins ubiquinated? – Wt vs ΔF508 ? Co-translational Ubiquitination of cystic fibrosis transmembrane conductance regulator in vitro regulator Past data shows rapid degradation of CFTR Past protein after translation protein – No Lag between synthesis and ubiquitination – Protein synthesis appeared to increase with Protein proteasome inhibitor proteasome – Use Hemin Paper suggest the opposite of convention theory Side Note: Techniques? Side NH2 HA tag COOH Protein Antibody Antibodies only bind specific sequences Can isolate protein of interest SDS­Polyacrylamide­gel electrophoresis SDS­Polyacrylamide­gel electrophoresis (SDS­PAGE) Western Blot (immuno blotting) Western Blot (immuno blotting) 2 1 Transfer Protein from gel (previous) on to a paper membrane (1,2) Now can use antibody to detect for presence of membrane (4) 3 4 Rough Microsomes (RM) Why was it beneficial to use a cell­free Why was it beneficial to use a cell­free translation system for this study? Why is hemin included in the experiments? General Methods General Rabbit Reticulum Lysate (RRL) - Cell lysate – provides all material need for translation - Just need to add mRNA of interest - Can now follow translation in vitro Can in What is the problem for this particular protein ? CFTR Rough Microsomes (RM) Rough Microsomes (RM) Serves as an artificial membrane for targeting and synthesis Now we can do experiment Fig 13-30, MBOC4 Figure 1. Purpose? Figure A. Diagram of Protein – - Note: location of glycosylation - What changes have been made? - HA tag allows for tracking of protein with an antibody B. First test systemC. Products of different size because of glcosylation (CHO). What must this mean? D. Another Negative control. Rules out nonspecific binding Figure 2. Purpose? Figure A. getting smear common A. to Ubiquitination to - both mutant and CFTR both are affected the same. are - supports previous hypothesis (75% / 90%) hypothesis - Hemin- what for ? Hemin B. Location of ubiquitin and B. protein protein Protein in RM only Both use radio labeled Both Ubiquitin (Ub) proteins. Figure 3. Purpose? Purpose? A. Timing of translation in A. proportion to Ub Ubiquitination happens early (20 min) (20 Full length protein not made Full till around 30 min till Ub added before –protein Ub synthesis is complete synthesis Figure 4. Purpose Figure Results of figure one could be Results because of premature release of ribosome ribosome Use mRNA without a stop codon Ribosome still translating mRNA Ubiquitination – co-translational Ubiquitination process process Ubq added – protein still attached Ubq to ribosome to Summary of Results Found protein ubiquitinated coFound translational Expect posttranslational Two possible explanations Summary of Results Summary 1. Early detection of mis­folded proteins 2. Ub has a chaperone type role ­ Ub aides in protein folding ­ Reversible by isopeptidase ­ Short chain no degradation ­ Long Chain degradation ...
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