The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Use 1µg for 106 cells. ab170190-Mouse monoclonal IgG1, is suitable for use as an isotype control with this antibody.
Use a concentration of 5 µg/ml.
1/5000. Detects a band of approximately 31 kDa (predicted molecular weight: 28 kDa).
Use at an assay dependent concentration. PubMed: 19960480
Use a concentration of 1 µg/ml.
Adapter protein implicated in the regulation of a large spectrum of both general and specialized signaling pathways. Binds to a large number of partners, usually by recognition of a phosphoserine or phosphothreonine motif. Binding generally results in the modulation of the activity of the binding partner. Negatively regulates the kinase activity of PDPK1.
Abundantly expressed in brain, heart and pancreas, and at lower levels in kidney and placenta. Up-regulated in the lumbar spinal cord from patients with sporadic amyotrophic lateral sclerosis (ALS) compared with controls, with highest levels of expression in individuals with predominant lower motor neuron involvement.
Belongs to the 14-3-3 family.
Ser-232 is probably phosphorylated by CK1.
Cytoplasm. In neurons, axonally transported to the nerve terminals.
Immunoprecipitation - Anti-14-3-3 Tau antibody [3B9] (ab10439)
14-3-3 Tau was immunoprecipitated using 0.5mg HeLa whole cell extract, 5µg of Mouse monoclonal to 14-3-3 Tau and 50µl of protein G magnetic beads (+). No antibody was added to the control (-).
The antibody was incubated under agitation with Protein G beads for 10min, HeLa whole cell extract lysate diluted in RIPA buffer was added to each sample and incubated for a further 10min under agitation.
Proteins were eluted by addition of 40µl SDS loading buffer and incubated for 10min at 70°C; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab10439.
Secondary: Goat polyclonal to mouse IgG light chain specific (HRP) at 1/20,000 dilution.
Band: 31kDa, non specific band - 26kDa: We are unsure as to the identity of this extra band; 14-3-3 Tau
Immunocytochemistry/ Immunofluorescence - Anti-14-3-3 Tau antibody [3B9] (ab10439)
ICC/IF image of ab10439 stained HeLa cells. The cells were 100% methanol fixed (5 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab10439, 1µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-mouse IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.
Western blot - Anti-14-3-3 Tau antibody [3B9] (ab10439)
All lanes : Anti-14-3-3 Tau antibody [3B9] (ab10439)
Lane 1 : MDA-MB-231 (Human breast adenocarcinoma cell line) Whole Cell Lysate Lane 2 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate Lane 3 : A431 (Human epithelial carcinoma cell line) Whole Cell Lysate Lane 4 : Jurkat (Human T cell lymphoblast-like cell line) Whole Cell Lysate Lane 5 : Y79 (Human retinoblastoma cell line) Whole Cell Lysate Lane 6 : NIH 3T3 (Mouse embryonic fibroblast cell line) Whole Cell Lysate
Lysates/proteins at 20 µg per lane.
Secondary Goat Anti-Mouse IgG H&L (HRP) preadsorbed (ab97040) at 1/5000 dilution Developed using the ECL technique
Flow Cytometry-Anti-14-3-3 Tau antibody [3B9](ab10439)
Overlay histogram showing SH-SY5Y cells stained with ab10439 (red line). The cells were fixed with 4% paraformaldehyde (10 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab10439, 1µg/1x106 cells) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 2µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in SH-SY5Y cells fixed with 80% methanol (5 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.
McFerrin MB et al. Dysregulation of 14-3-3 proteins in neurodegenerative diseases with Lewy body or Alzheimer pathology. Ann Clin Transl Neurol4:466-477 (2017).
Read more (PubMed: 28695147) »
Broadbelt KG et al. Brainstem deficiency of the 14-3-3 regulator of serotonin synthesis: a proteomics analysis in the sudden infant death syndrome. Mol Cell Proteomics : (2011).
Read more (PubMed: 21976671) »