Cell immunostaining was performed as previously described with slight modification [25 (link),26 (link)]. Briefly, N2A−Htau cells were plated on Matrigel-coated 35 × 10 mm No 1.5 glass-bottom dishes (Eppendorf) and grown to 60% confluence prior to VPS35 siRNA transfection. After a 72 h incubation, cells were washed with PBS and fixed in 4% paraformaldehyde in PBS for 20 min at RT. After washing several times with PBS and permeabilizing with 0.2% Triton X-100 in PBS, cells were incubated in blocking solution (5% normal donkey serum in PBS) for 1 h at RT followed by an O/N incubation at 4 °C with a combination of primary antibodies prepared in 2% blocking solution in PBS against VPS35 (1:250, Abcam), Tau13 (1:200, Biolegend), PHF13 (1:20, Thermo), AT270 (1:100, Thermo), and MC-1 (1:20, provided by Peter Davies). After several washings with PBS, cells were incubated for 1 h at RT with secondary Alexa Fluor 568-conjugated antibody against VPS35 and 647-conjugated antibody against Tau13, AT270, PHF13 or MC-1 (all at 1:250; Abcam). Cells were then stained with DAPI, washed with PBS and subsequently held at 4 °C in the dark until imaging.
Tau13
Manufactured by BioLegend
Sourced in United States
Tau13 is a monoclonal antibody produced by BioLegend. It is designed for the detection of the Tau protein, which is a microtubule-associated protein involved in the stabilization of microtubules within cells. The Tau13 antibody can be used in various research applications, such as Western blotting, immunohistochemistry, and immunocytochemistry, to study the expression and localization of the Tau protein.
Automatically generated - may contain errors
Lab products found in correlation
Phf13, by Thermo Fisher Scientific (2 mentions)
No 1.5 glass bottom dishes, by Eppendorf (2 mentions)
Alexa fluor 568 conjugated antibody against vps35, by Abcam (2 mentions)
647 conjugated antibody against tau13, by Abcam (2 mentions)
At270, by Abcam (2 mentions)
Phf13, by Abcam (2 mentions)
Vps35, by Abcam (2 mentions)
At270, by Thermo Fisher Scientific (2 mentions)
Gapdh, by Merck Group (1 mentions)
Gapdh, by GE Healthcare (1 mentions)
Ecl plus, by GE Healthcare (1 mentions)
Nupage 4 12 bis tris gel, by Thermo Fisher Scientific (1 mentions)
Hrp conjugated anti rabbit igg, by GE Healthcare (1 mentions)
Gapdh, by Meridian Bioscience (1 mentions)
Cf633, by Biotium (1 mentions)
Polyvinylidene fluoride pvdf membrane, by Thermo Fisher Scientific (1 mentions)
At 270 pt181, by Thermo Fisher Scientific (1 mentions)
6 protocols using tau13
1
Immunostaining of Tau Protein Variants
2
Musashi Protein Oligomerization in Alzheimer's Disease
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Western blot analyses were performed with both recombinant Musashi proteins and homogenates from AD and age-matched control brain tissues. Approximately 4 μg of Musashi oligomeric preparations and 10 μg of each brain homogenate were loaded on precast NuPAGE 4–12% Bis-Tris gels (Invitrogen) for SDS-PAGE analyses. Gels were subsequently transferred onto nitrocellulose membranes and blocked overnight at 4 °C with 10% nonfat dry milk. Membranes were then probed for 1 h at room temperature with α-Oligomeric antibody F11G3 (1:1000), α-MSI1 (1:1000, Abcam), α-MSI2 (1:1000, Abcam) and Tau13 (1:10,000, BioLegend), GAPDH (1:1000, Sigma) antibodies diluted in 5% nonfat dry milk. α-Oligomeric antibody F11G3, α- MSI1 and α- MSI2 immunoreactivity were detected with an HRP-conjugated anti-rabbit IgG (1:6000, GE Healthcare). Tau13 and GAPDH immunoreactivity were detected using an anti-mouse IgG (1:6000, GE Healthcare) diluted in 5% milk. ECL plus (GE Healthcare) was used to visualize the bands.
3
Western Blot Protocol for Protein Detection
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Nitrocellulose membranes were blocked in a 5% powdered milk (Nestle Carnation, Glendale, CA) solution in PBS-T. The membrane was then incubated in primary antibody in the blocking solution overnight, followed by three 10-min washes in PBS-T. Primary antibodies included GAPDH (1:5000, Meridian Life Science, Memphis, TN), Tau13 (1:1000, BioLegend, San Diego, CA), mouse/human SOD1 (1:4000, generated in-house) and human SOD1 (1:2500, generated in-house [55 (link)]). Horseradish peroxidase-conjugated secondary anti-mouse or anti-rabbit antibody (Vector Laboratories, Burlingame, CA) was used in order to visualize proteins by chemiluminescence using the Pierce ECL Western Blot Substrate Kit (Thermo Scientific, Waltham, MA). Western blot quantification was conducted using GeneTools by Syngene (in correlation with the GeneSys imager, Daly City, CA).
4
Fluorescent Tau13 Antibody Western Blot
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For western blots, a fluorescent anti-tau antibody was used. Therefore tau13 (Biolegend, San Diego, USA) was labelled with CF633 (Biotium, Freemont, USA), and the labelling process was performed as described previously [37 (link)]. 2N4R tau recombinant protein samples were prepared in Laemmli buffer (final 1× composition: 20 mM Tris, pH 6.8, 2% SDS, 6% glycerol, 1% β-ME, 0.002% Bromophenol Blue). All samples were heated at 95°C for 5 min and separated using SDS PAGE (15%). Proteins were then transferred to a polyvinylidene fluoride (PVDF) membrane (Thermo Fisher Scientific, Waltham, USA) at 500 mA for 40 min. After a washing step for 15 min in Tris-buffered saline tween buffer (TBS-T) (20 mM Tris, 150 mM NaCl, 0.1% Tween 20), the membrane was blocked for 1 h with 2.5% milk powder/TBS-T. Next, the membrane was washed with TBS-T, 2 × 5 min and in the last step for 15 min. tau13 stocks were 1 mg/ml and were diluted in TBS-T (1:5000). The membrane was incubated with the antibody for 1.5 h. at RT. After a final wash step (2 × 5 min and 1 × 10 min), TBS-T was performed. Detection based on the CF633 fluorescence of the labelled tau13 antibody. Bio-Rad universal hood II and Chemidoc XRS camera and Quantity One 4.6.5 software enabled the visualisation and quantification of the protein bands.
5
Immunostaining of Tau Protein Variants
6
Tau Protein Phosphorylation Analysis
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BT‐2 (RRID: AB_10975238), AT8 (RRID: AB_223647), and AT‐270 (pT181) (RRID: AB_223651) were from Thermo Fisher Scientific, Tau‐13 (RRID: AB_291452) was from Biolegend, pT175 (Cat: MM‐0147‐P) was from MÉDIMABS, and Cell Signaling Technology developed pT217 (E9Y4S), pT231 (E7O3C), and pTPP (D61C3).
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