Proteins were electrically transferred to a polyvinylidene difluoride (PVDF) membrane at 100 Volts for 1 h. The total protein content of the crude extracts was estimated by Ponceau staining prior to immunodetection. The PVDF membrane was then incubated for 1 h at room temperature with 5% bovine serum albumin in Tris-buffered saline (TBS) at pH 7.5 and 0.5% tween-20 (TBS-T). Subsequently, the membrane was incubated overnight at 4 °C with a 1:500 dilution in TBS-T of the monoclonal W. anomalus ATCC 96603 KT-neutralizing antibody mAbKT4 , known to cross-react with KTs from other Wickerhamomyces  and Williopsis  strains. After washing three times in TBS-T, the membrane was incubated for 1 h at room temperature with a secondary, peroxidase-conjugated anti-mouse antibody. The membrane was thoroughly washed with TBS-T, incubated for 1 min with the proper substrate (BM Chemiluminescence blotting substrate, Roche, Basel, Switzerland), and detected by ChemiDoc 2000R (Kodak, Rochester, NY, USA).