1. Yeast Secretory Pathway Lab 3: Probing & Developing the Western Blot Your blots have been incubating in blocking buffer (5% milk in PBS with 0.25% Tween) for 1 hour. Pour off blocking buffer and add 10 ml of primary antibody (rabbit polyclonal anti-pre-pro- alpha factor) diluted 1:2000 in blocking buffer (already diluted for you). Incubate at RT with shaking for 1 hour.

2. Predicting Your Results How do you expect the size of the PPAF to differ among the various samples? Wildtype 25°C: 37°C sec18 25°C 37°C sec61 25°C 37°C

3. Sample Blot Translated MF  1 protein product = 18.6 kDa Fully glycosylated PPAF = 26 kDa (N-linked glycosylation at 3 sites)

4. ECL: Enhanced Chemiluminescence Luminol is oxidized and gives off light Detection reagent supplier: Amersham Biosciences (ECL Western Detection Reagent)

5. Next steps: wash, 2° Ab, wash Pour off the primary antibody into the sink (no need to save). Wash blots 3 x 10 minutes at RT in PBST (phosphate buffered saline, pH 7.4 w/ 0.25% Tween) on shaker. After the last wash, remove as much buffer as possible by tapping edge of container on towel. Add diluted (1:5000) secondary antibody (HRP-conjugated goat anti-rabbit antibody) to the nitrocellulose membrane. Incubate 30 minutes at RT with shaking. Wash 3 x 10 minutes with PBST. Wait until your instructor is ready to help you with developing before pouring off the last wash solution.

6. ECL Development Protocol Mix 1 ml each of ECL detection reagents 1 & 2 in a small glass test tube (use separate pipets to measure!). Vortex to mix. Place nitrocellulose membrane on plastic wrap and cover with detection reagent for 1 min. Touch edge of nitrocellulose to paper towel to remove liquid. (Handle membrane with forceps.) Place membrane (protein side up) in autoradiography cassette between page protectors. Go to dark room with instructor.

7. ECL in darkroom Activate Stratagene ™ logo with light (20 sec) Make sure all lights are off except safe light and place film on top of blot without moving it around. Close cassette. Wait 1 minute and remove film. (May need to repeat w/ longer or shorter exposure time.) Feed film into developer—processing takes 4 minutes. Line up developed film with your nitrocellulose membrane blot using Stratagene™ logo marker so that you can mark the MW standards on the film using a Sharpie. Use a gel comb to you help find lanes.

8. Notes on the Yeast Lab Report Worth 45 points; same sections as for first report. See grading rubric in folder on the lab conference. Pay attention to comments on first report. See me or consult posted sample report (Lab Report Info folder) for clarification. Due April 9 (Mon. lab) or April 12 (Thurs. lab) by midnight. (E- mail submission acceptable.)

9. Notes on the Yeast Lab Report Central question: What steps in the yeast secretory pathway are carried out by Sec18 & Sec61? (Approach the paper acknowledging that the roles of these proteins have been previously determined by others, but that you have used two experimental methods to confirm their results.) Intro should include previously determined roles for Sec18 & Sec 61, outline of methods we used to analyze the sec18 & sec61 mutants, your hypotheses for the experimental outcomes of the two methods & a brief statement of the actual conclusions you drew from your experiments. M&M should include complete genotypes of all yeast strains (including mating type—all strains are MAT  ) & descriptions of all plasmids. Should cite sources of yeast strains, plasmids, antibodies & ECL developing kit (see posting on conference). Can cite lab manual for “standard methods” for gel & Western blot. Discussion should address any discrepancies between results & hypotheses & should relate your data to previously published work on Sec18 & Sec61. * Consult annotated version of Deshaies & Schekman (1987) posted in Referernces folder on lab conference for more help with appropriate style for various sections.