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-proalpha 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 MF1 protein product = 18.6 kDa
Fully 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. (Email 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.