Antiha 12c5

Samples for quantification of biotinylation were allowed to cool for at least 10 minutes after boiling. 6 ul of boiled sample was combined with 1.5 ul streptavidin (Invitrogen #434302 at 10 ug/uL), vortexed briefly, and loaded into an Invitrogen SDS-PAGE 1.5 mm 4–12% Bis-Tris gel. Gels were run for 2 hours at 4°C and 110V in 1x MOPS running buffer. Transfer was performed onto nitrocellulose membrane using a semi-dry transfer apparatus (Bio-Rad Turbo Blot). Membranes were blocked for 30 minutes in 5% milk in TBST and then incubated with mouse anti-HA primary antibody (antiHA-12C5 Roche #11583816001) in 5% milk in TBST with gentle shaking overnight at 4°C. After primary antibody incubation, membranes were washed three times in TBST for a total of 15 minutes and incubated at room temperature with IRDye 800CW Goat anti-Mouse IgG Secondary Antibody (1:5000, Licor #926–32210) in 5% milk in TBST for 1 hour. Blots were imaged on a Licor gel imager and band intensities were quantified using ImageStudioLite. All ratiometric analyses are the intensity of the top band divided by the intensity of the bottom band plus the intensity of the top band in a given lane.