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.