Tau 5 antibody

DNA from calf thymus (activated type XV), unfractionated whole histone from calf thymus (type II-A), poly-L-Lysine, heparin and hepes were from Sigma (St. Louis, MO, USA). SPR gold sensorchips were from Xantec Bioanalytics (Dusseldorf, Germany). PHF-1 antibody was kindly donated by Dr. Davies (Albert Einstein College of Medicine, NY, USA). According to residue numbering of the longest human Tau isoform (441 aa), PHF-1 antibody react with Tau when serines 396 and 404 are phosphorylated. Total tau protein was detected with Tau-5 antibody from Abcam (Cambridge, MA, USA). Unless otherwise indicated, all experiments were carried out in a neutral running buffer composed of 10 mM hepes, 0.1 M NaCl, pH 7.

To determine whether tau interacts with ERα, Co-IP analysis was conducted, using cortical protein lysates from postmortem brain tissues of AD patients and matched control subjects. Samples of frozen cortical gray matter of AD and control were homogenized in RIPA buffer containing protease inhibitors (Cell Signaling) on ice and centrifuged for 10 min at 16,000 × g at 4 °C. 500 ug of lysate was incubated with 20 ul of tau-5 antibody (Abcam) using Dynabeads® Protein G—CoIP Kit (Invitrogen) following the manufacturer’s protocol. Negative controls included IP with normal mouse IgG (Cell Signal). Input and IP samples were run on SDS-PAGE and western blot was performed using the ERα antibody and tau-5.