Purified anti ha 11 epitope tag antibody

The threshold sensors with either nuclear localization signal or nuclear export signal and a C-terminal 3HA tag were expressed from a pRS315 vector under the ADH1 promoter. Cells were grown in synthetic complete media lacking leucine (SC-LEU) with 2% glucose at 30 °C to OD 0.3. Cells were then treated for 2.5 hours with 1 µg/ml α-factor, washed thoroughly and released into fresh SC-LEU medium. 5 ml of cells were centrifuged every 10 minutes and flash-frozen in liquid N₂. Cells were lysed by bead beating in lysis buffer containing urea. Proteins were separated using Phos-tag SDS-PAGE with 50 µM Phos-tag and 8% acrylamide. Blotting of Phos-tag SDS-PAGE gels was performed using a dry system iBlot (Invitrogen). Purified anti-HA.11 epitope tag antibody (1:500) (clone 16B12, Biolegend) and HRP-conjugated anti-mouse antibody (1:7500) from Labas, Estonia were used to detect the 3HA-tagged proteins.

Samples were run on an 8% SDS-PAGE gel, and transferred to PVDF (BioRad). Blots were blocked, incubated overnight with 1:1000 primary antibody, washed and incubated with 1:4000 secondary antibody. HA was detected with Purified anti-HA.11 Epitope Tag antibody (16B12, BioLegend) and Goat Anti-Mouse HRP Conjugate antibody (cat# 170-5047, BioRad). Tubulin was detected with Rat Anti-Tubulin Alpha antibody (MCA78G, BioRad) and HRP-Goat Anti-Rat IgG(H+L) (cat# 62-9520, Invitrogen). Blots were developed using Pierce ECL Western Blotting Substrate on a G:BOX developer (Syngene). Each experiment was performed at least three times.