Verifying and qualifying secreted PvPGIP protein presence via
western blot
To verify that our PvPGIP protein was being secreted, samples were
prepared by using the protein secretion assay as follows. S.
cerevisiae strain PJ69-4A, was transformed containing either the
plasmid with full length PvPGIP1 with the HA tag (PvPGIP1-HA), or the
full length PvPGIP1 with the HA tag and preOST1 secretion signal
(preOST1-PvPGIP1-HA). An empty vector yeast was used as a negative
control. The yeast was grown for 72 hours in 2mL of -T YNB in a shaker
at 30°C at 250 rpm. 200µL of media was pipetted into 1.5mL tubes and
10µL of 1mg/ML BSA was added to each tube. 20µL of 0.15% deoxycholate
was added and samples were immediately vortexed then sat at room
temperature for 10 minutes. 20µL of 1M TCA was added to each sample,
vortexed, then placed on ice for 30 minutes to precipitate. Samples were
centrifuged for 30 minutes at 4°C at 22,000g. After centrifugation, the
supernatant was aspirated out and resultant pellet was washed with 200µL
of ice-cold acetone once. Pellet was left to air dry at room temperature
for 30 minutes, then was treated with 32.5µL DI water, 12.5µL of LDS
buffer, and 5µL of reducing agent. Samples were mixed by pipetting and
heated to 70°C for ten minutes. After heating, the tubes were
centrifuged at room temperature for five minutes and supernatant was
used to load into a gel. An 8% tris-glycine gel was used for the
SDS-PAGE which was made according to the protocol found in the Surecast
Handcast System Quick Reference sheet by Thermo Fisher Scientific. The
SDS-PAGE was run at 150V for one hour in tris-glycine running buffer
with a ladder (Fisher Scientific, catalog #50491514). A protein
transfer with a PVDF membrane (Fisher Scientific, catalog #ISEQ0010)
followed using the Protein Transfer Technical Handbook provided by
Thermo Fisher Scientific. Once finished, the membrane was removed and
incubated at room temperature for 2 hours in 1x TBST + 5% milk. This
was washed twice with 1x TBST for 5 minutes each time, then incubated in
10mL of 1x TBST + 3% milk + 2µL of antibody (Anti-HA tag antibody HRP
ab1190 from Abcam) overnight at 4°C. A standard protein detection
protocol was then used according to the Protein Detection Technical
Handbook from Thermo Fisher Scientific.