BIOL110L-12-Module 1-Data Preparation Oral presentations: Come prepared to describe your data (using lab terminology) and to provide interpretations of the data. The presentation is informal; the purpose is to start preparing to write Lab Report #1. You ’ ll need to prepare three figures using the format/legend indicated: Figure 1 : Show drawings of cells from morphology experiments. Show drawings of one or two intact yeast per experiment: in log and saturated phase of growth, spheroplasts in iso- osmotic buffer, high salt buffer, low salt buffer, in 2% Triton X100, in 2% SDS and in 15% hexandiol. Figure 2 : Show (4) photos (light and fluorescent) of your yeast examined under the fluorescent microscope, with and without 15% 1,6 hexanediol. Make an assignment of what compartment your protein resides in, and what the effect of hexanediol was on your proteins localization. Figure 3 : Show the amido black scanned gel as labeled (A) . Show the Western blot results as labeled (B). Come prepared to explain your interpretation of the results and what can be learned regarding the cellular location of the unknown GFP fusion.
Figure 1 Morphological observation of yeast * Know reason behind each morphological change* Place your drawings here 1. Intact yeast in log phase of growth >> 2. Intact yeast in saturated phase of growth >> 3. Yeast spheroplasts in iso-osmotic buffer >> 4. Yeast spheroplasts in high salt buffer >> 5. Yeast spheroplasts in low salt buffer >> 6. Yeast spheroplasts in 2% Triton X-100 >> 7. Yeast spheroplasts in 1% SDS >> 8. Yeast spheropalsts in 15% 1,6 hexanediol >>
Yeast strain YS2 expressing Ule1-GFP Figure 2 Log phase growth minimal media with 1,6 hexanediol minimal media Light microscopy Fluorescence microscopy “ Shown are yeast expressing a fusion between GFP and Ule1 proteins, visualized under the light microscope using a 100x objective; *Discuss your results on protein localization For example: “ Ule1-GFP is an abundant cytosolic protein, excluded from vacuoles and possibly even the cell nucleus (how can you tell?) Ule1 is a GTPase activating protein that accelerates GTP hydrolysis of Elu1 GTPase in the cytoplasm
Discuss your results and whether they match your expectation based on what Figure 3 you know about the protein ’ s MW (from SGD) A B Western blot staining Amido black staining with anti-GFP antibodies (detection of all proteins) (detection of your GFP protein) MSS + 15% 1,6 HD MSS + 15% 1,6 HD MSS + 2% TX-100 MSS + 2% TX-100 MSS + 1M NaCl MSS + 1M NaCl MSS + buffer MSS + buffer WCE MSP WCE HSP HSS LSP MSP HSP MW HSS LSP MW kDa kDa 200 200 116 116 97 97 68 68 44 44 30 30 Expected molecular weight of Ule1 + GFP is: 50 kDa (Ule1) + 26 kDa (GFP) = 76 kDa for the fusion Does it match the apparent molecular weight of your protein by Western?
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