Anti kss1

Yeast lysates were fractionated by either conventional SDS-PAGE or Mn²⁺ Phos-tag SDS-PAGE before transfer to polyvinylidene difluoride (PVDF) membranes. Membranes were blocked with 5% powdered milk in Tris-buffered saline solution with 0.05% Tween-20 (TBST) for 2 hr at room temperature and incubated with primary antibodies diluted 1:2000 in TBST containing 5% milk overnight at 4 °C. Primary antibodies included: anti-Penta-His (QIAGEN, #34650), anti-FLAG M2 (Sigma-Aldrich, #F3165), and anti-Kss1 (Santa Cruz, #sc-6775-R). Membranes were washed with TBST before incubation with fluorophore-labeled secondary antibodies (1:20,000 dilution) in TBST containing 5% bovine serum albumin (BSA) for 1 hr at room temperature. Membranes were washed with TBST and imaged with an Odyssey CLx imager (LI-COR Biosciences). Fluorescence signals were quantified using Image Studio software.

Yeast lysates were fractionated by either conventional SDS-PAGE or Mn²⁺ Phos-tag SDS-PAGE before transfer to polyvinylidene difluoride (PVDF) membranes. Phos-tag acrylamide gels were prepared with 12% acrylamide, 100 µM MnCl₂, and 50 µM Phos-tag Acrylamide (Fujifilm AAL-107). Membranes were blocked with 5% powdered milk in Tris-buffered saline solution with 0.05% Tween-20 (TBST) for 2 h at room temperature and incubated with primary antibodies diluted 1:2000 in TBST containing 5% milk overnight at 4 °C. Primary antibodies included: anti-Penta-His (QIAGEN, #34650), anti-FLAG M2 (Sigma-Aldrich, #F3165), and anti-Kss1 (Santa Cruz, #sc-6775-R). Membranes were washed with TBST before incubation with fluorophore-labeled anti-mouse IgG (LI-COR Biosciences, #D10603-05) or anti-rabbit IgG (Invitrogen, #A21109) secondary antibodies at 1:20,000 dilution in TBST containing 5% bovine serum albumin (BSA) for 1 h at room temperature. Membranes were washed with TBST and imaged with an Odyssey CLx imager (LI-COR Biosciences). Fluorescence signals were quantified using Image Studio 5.2.5 software.