BIOL110L-12-Module 1-Data Preparation Oral presentations: Come - - PowerPoint PPT Presentation

▶

Nov 29, 2022 291 likes •341 views

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

SLIDE 1

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-

smotic 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.

SLIDE 2

Figure 1

Morphological observation of yeast

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 >>

Place your drawings here

* Know reason behind each morphological change*

SLIDE 3

Figure 2 Yeast strain YS2 expressing Ule1-GFP

Light microscopy Fluorescence microscopy Log phase growth

“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

minimal media minimal media with 1,6 hexanediol

SLIDE 4

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?

Figure 3 Western blot staining with anti-GFP antibodies

(detection of your GFP protein)

kDa 200 116 97 68 44 30 MW WCE LSP MSP HSS MSS + buffer

Amido black staining

(detection of all proteins)

MSS + 1M NaCl MSS + 2% TX-100 kDa 200 116 97 68 44 30

B A

HSP MSS + 15% 1,6 HD MW WCE LSP MSP HSS MSS + buffer MSS + 1M NaCl MSS + 2% TX-100 HSP Discuss your results and whether they match your expectation based on what you know about the protein’s MW (from SGD) MSS + 15% 1,6 HD