Immunofluorescence analysis of Tau [pS199] was done on 70% confluent log phase U-2 OS cells. The cells were fixed with 4% paraformaldehyde for 15 minutes, permeabilized with 0.25% Triton™ X-100 for 10 minutes, and blocked with 5% BSA for 1 hour at room temperature. The cells were labeled with ABfinity™ Tau [pS199] Recombinant Rabbit Monoclonal Antibody (701054) at a dilution of 1:500 in 1% BSA and incubated for 3 hours at room temperature and then labeled with Alexa Flour® 488 Goat Anti-Rabbit IgG Secondary Antibody (A11008) at a dilution of 1:400 for 30 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with SlowFade® Gold Antifade Mountant with DAPI (S36938). F-actin (Panel c: red) was stained with Alexa Fluor® 594 Phalloidin (A12381) and Panel d is a merged image showing nuclear and cytoplasmic localization and panel e shows competition with the phospho-Tau[pS199] peptide. The images were captured using a Nikon microscope at 20X magnification.
|Tested species reactivity||Human, Mouse, Rat|
|Host / Isotype||Rabbit / IgG|
|Immunogen||Phosphopeptide corresponding to amino acids 193–203 of human microtubule-associated protein tau|
|Contains||0.09% sodium azide|
|Storage Conditions||Maintain refrigerated at 2-8°C for up to 1 month. For long term storage store at -20°C|
|Tested Applications||Dilution *|
|Flow Cytometry (Flow)||1:100-1:500|
|Immunohistochemistry (Paraffin) (IHC (P))||1:50-1:500|
|Western Blot (WB)||1:500-1:5000|
* Suggested working dilutions are given as a guide only. It is recommended that the user titrate the product for use in their own experiment using appropriate negative and positive controls.
This antibody is predicted to react with human based on sequence homology.
ABfinity™ recombinant antibodies are rabbit monoclonal antibodies, unmatched for producing superior results. ABfinity™ antibodies are developed by immunizing animals, screening for functionality, cloning the immunogen-specific antibody genes into high-level mammalian expression vectors, produced on a large scale, and purified with Protein A.
ABfinity™ monoclonal antibodies resemble rabbit monoclonals isolated from serum or produced by hybridomas, but demonstrate greater specificity and sensitivity. Because ABfinity™ recombinant antibodies are derived from cloned DNA sequences of the heavy and light antibody chains, they are not susceptible to cell-line drift or lot-to-lot variation, thus allowing for peak specificity and performance.
Intact IgG appears on a non-reducing gel as ~150 kDa band and upon reduction generating a ~25 kDa light chain band and a ~50 kDa heavy chain.
Tau protein plays a very important role in nucleation and stabilization of microtubules by associating with tubulin subunits. In humans, Tau is predominantly expressed in the brain, and is involved in neurodegeneration when the protein is misfolded, hyperphosphorylated, or self-assembles into tangles. Six isoforms of Tau with varying degree of affinity to tubulin exist. Numerous serine/threonine kinases, including GSK-3 beta, protein kinase A, and casein kinase II (CK2) phosphorylate Tau. Phosphorylation of the serine 396 residue is triggered through GSK-3beta and cdk5. The gene encoding the Tau protein consists of 16 exons, and is located at chromosomal locus 17q21.
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