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.

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

Sample Blot

Translated MF1 protein product = 18.6 kDaFully glycosylated PPAF = 26 kDa(N-linked glycosylation at 3 sites)

ECL: Enhanced Chemiluminescence

Luminol is oxidized and gives off light

Detection reagent supplier:Amersham Biosciences(ECL Western Detection Reagent)

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.

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.

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.

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.)

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.