Publication protocol
For sarkosyl-insoluble fractions, total protein concentration was normalized according to pellet weights. Total protein concentrations for all other samples were determined by Pierce™ Bicinchoninic Acid protein assay (Thermo Scientific). Equal amounts of protein for each sample were loaded and separated using SDS-PAGE on 10%, 10.5–14%, or 10–20% tris-HCl gels (Bio Rad). Protein was transferred to nitrocellulose membranes (Bio Rad), which were blocked with 5% bovine serum albumin (Sigma) in 1× tris buffered saline buffer with Tween 20 (TBST) buffer (10 mM Tris-Base (Sigma), 0.2 M NaCl (Macron Chemicals), 0.1% Tween-20 (Sigma) pH 7.4). Protein was immunoblotted with Tau46 (Cell Signaling Technology #4019, dilution 1:10,000), Tau13 (BioLegend #MMS-520R, dilution 1:60,000), GAPDH(14C10) (Cell Signaling Technology #2118, dilution 1:4000), GAPDH(GA1R) (Thermo Scientific #MA5-15738, dilution 1:5000), AT8 (Thermo Scientific #MN1020, dilution 1:1000), anti-human tau (Abcam # ab74391, dilution 1:10,000), and βIII-tubulin (ProSci #79–720, dilution 1:10,000) antibodies. Additional antibodies from Peter Davies for phospho-epitopes on tau included MC1 (dilution 1:800), CP13 (dilution 1:1,000), and PHF1 (dilution 1:1,500). To visualize antibody immunoreactivity using a LiCor imaging system and Image Studio software, IRDye-linked goat anti-mouse 800CW and goat anti-rabbit 680LT secondary antibodies were used (LI-COR Biosciences,dilution 1:100,000). Following LiCor image acquisition using Image Studio software (Odyssey), Amido black staining solution (Sigma-Aldrich) was used for total protein quantification according to manufacturer’s instructions. Immunoreactivity and Amido black staining were quantified by densitometry using OptiQuant version 3 software, following guidelines for total protein quantification.
Full paper
Login or
join for free to view the full paper.
Videos
Check out videos that might be relevant for performing Western blotting Tau using Phospho-Tau (Ser202, Thr205) Monoclonal Antibody (AT8) from Thermo Fisher Scientific. Please note that these videos are representative and steps or experiment specific processes must be kept in mind to expect desired results.
We haven't found any additional videos for this experiment / product combination yet.