Pvdf immunoblot membrane

Protein was extracted 48 hours after doxycycline treatment using M-PER lysis buffer (Thermo) supplemented with 1% phosphatase and protease inhibitors (Thermo). Protein concentrations were determined using BCA assay (Thermo) on an Optima plate reader (BMG Labtech). Proteins were then resolved using 10% Tris-gycine gels (Thermo). After separation, protein (50 µg) was transferred to a PVDF-ImmunoBlot membrane (BioRad) and blocked in 5% non-fat milk for 1 hour at room temperature on a rocking platform. The blots were then incubated with primary antibody overnight at 4°C on a rocking platform. Antibodies were applied as follows: sheep anti-human SIAE (Custom, see antibody production) 3 ug/mL and mouse anti-human β-actin (Sigma). Subsequently, the blots were washed five times in Tris-buffered saline containing 0.05% Tween-20 before and after 1 hour of incubation at room temperature with Licor 800CW infared secondary antibodies. Blots were performed in dupicate. β-actin was used to control for protein loading. Blots were imaged by Licor Odyssey Clx technology using Image Studio 5 software.

In order to confirm recombinant proteins were correctly sized, SDS PAGE was performed using a BioRad gel electrophoresis system. Samples containing recombinant EK-PSA-GZMB/TRP proteins were mixed 1:1 with 4X reducing SDS PAGE Sample Buffer and boiled for 5 minutes. Samples were then loaded and run at 150V until fully migrated. For protein staining, the gel was then washed 2-3 times with RO H₂O and stained for one hour with SimplyBlue Safe Stain solution (Thermo LC6060). To de-stain samples were washed in RO H₂O overnight. For Western blotting, samples were run on a non-reducing gel and were transferred to PVDF Immuno-Blot membrane (BioRad 1620177) for 1 hour at 100V and incubated for one hour in TBST containing 5% milk. To monitor removal of the DDDK peptide, an anti-DDDDK polyclonal antibody (Abcam 1162) was diluted to 1:5000 in TBST/milk and incubated with the membrane overnight at 4°C. The next day, the membrane was washed 5X with TBST and incubated with an anti-rabbit HRP-linked IgG (Cell Signaling #70762) diluted at 1:10000 in TBST/milk for one hour at 4°C. The blot was then washed 5X with TBST and incubated with chemiluminescent substrate solution (Thermo 34077) diluted according to manufacturer's instructions. The blots were then developed at specified time points.