Cdp star substrate

Sensitive human tau and βIII tubulin ELISAs were adapted and modified according to previous reports (Barten et al., 2011 (link), Meredith et al., 2013 (link)). Briefly, mouse monoclonal antibody HT7 or rabbit monoclonal βIII tubulin antibody (ab68193; Abcam) was used for capture. The respective analytes were detected with alkaline phosphatase-conjugated mouse monoclonal antibodies Tau5 or TUJ1 (806401, 801201; BioLegend). Recombinant full-length human tau (rPeptide) and recombinant βIII tubulin (Cytoskeleton) were used to generated standard curves for each assay. The CDP-Star substrate (T2214, Invitrogen) was used as a chemiluminescent alkaline phosphatase substrate. See Supplemental Experimental Procedures for detailed methods.

Cells were grown to early stationary phase under continuous illumination (8 μE m^–2 s^–1) in agitated 200 ml liquid cultures in Tris-acetate-phosphate (TAP) medium. Genomic DNA extraction and Southern blot were performed as in (57 (link)), except that instead of radioactive probes, oligonucleotide probes biotinylated at both ends were used (Sultan_oZX076: 5′-GGCTGCGTGGCTGGACTGCTGCACT-3′, Suber_oZX179: 5′-GCCACAGGGGAAAGTCAGAGAATCTG-3′, rDNA_oZX178: 5′-ACGCGCATGCACTCACAGCACGTCA-3′ and telomere_oT959: 5′-CTAAAACCCTAAAACCCTAAAACCCTAAAAC-3′; Eurofins Genomics) and detected by chemiluminescence. After hybridization of the probe, the membrane was washed 3×5 min in wash buffer (58 mM Na₂HPO₄, 17 mM NaH₂PO₄, 68 mM NaCl, 0.1% SDS). The membrane was next processed for detection with three successive incubations (5, 5 and 30 min) in blocking buffer (Thermo Scientific, Nucleic Acid Detection Blocking Buffer) before a 30 min incubation with alkaline phosphatase-conjugated streptavidin (Invitrogen) diluted in blocking buffer (0.4 μg/ml). The membrane was then washed again 3 × 5 min in wash buffer, incubated 2 × 2 min in assay buffer (0.1 M Tris, 0.1 M NaCl pH9.5) and 5 min in CDP-Star substrate (Applied Biosystems) before imaging with a G:BOX Gel Doc system (Syngene).