Western blot analysis of extracts from various mouse, human, and rat brain tissues using Tau 4R Antibody (upper), or β-Actin (D6A8) Rabbit mAb #8457 (lower). Mouse hTau-KO mouse brain tissue was kindly provided by Dr. Dominic Walsh at Brigham and Women's Hospital and Harvard Medical School.
Western blot analysis of extracts from 293T cells, untransfected (-), or transfected with Tau isoforms 2, 4, 5 (-), 6, 7, or 8(+) using Tau 4R Antibody (upper). Tau (D1M9X) XP® Rabbit mAb #46687 was used as a control (lower).
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Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA and 50% glycerol. Store at –20°C. Do not aliquot the antibody.
For western blots, incubate membrane with diluted primary antibody in 5% w/v BSA, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight.
NOTE: Please refer to primary antibody datasheet or product webpage for recommended antibody dilution.
From sample preparation to detection, the reagents you need for your Western Blot are now in one convenient kit: #12957 Western Blotting Application Solutions Kit
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
Load 20 µl onto SDS-PAGE gel (10 cm x 10 cm).
NOTE: Volumes are for 10 cm x 10 cm (100 cm2) of membrane; for different sized membranes, adjust volumes accordingly.
* Avoid repeated exposure to skin.
posted June 2005
revised November 2013
Reprobing of an existing membrane is a convenient means to immunoblot for multiple proteins independently when only a limited amount of sample is available. It should be noted that for the best possible results a fresh blot is always recommended. Reprobing can be a valuable method but with each reprobing of a blot there is potential for increased background signal. Additionally, it is recommended that you verify the removal of the first antibody complex prior to reprobing so that signal attributed to binding of the new antibody is not leftover signal from the first immunoblotting experiment. This can be done by re-exposing the blot to ECL reagents and making sure there is no signal prior to adding the next primary antibody.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalently purified water.
posted June 2005
revised October 2016
Protocol Id: 10
Tau 4R Antibody recognizes endogenous levels of total tau isoforms 6, 7, and 8 protein. This antibody does not cross-react with tau isoforms 2, 4, or 5.Species Reactivity:
Human, Mouse, Rat
Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues surrounding amino acid Gly273 of isoform 8 of human tau protein. Antibodies are purified by protein A and peptide affinity chromatography.
Tau is a heterogeneous microtubule-associated protein that promotes and stabilizes microtubule assembly, especially in axons. Six isoforms with different amino-terminal inserts and different numbers of tandem repeats near the carboxy terminus have been identified, and tau is hyperphosphorylated at approximately 25 sites by Erk, GSK-3, and CDK5 (1,2). Phosphorylation decreases the ability of tau to bind to microtubules. Neurofibrillary tangles are a major hallmark of Alzheimer's disease; these tangles are bundles of paired helical filaments composed of hyperphosphorylated tau. In particular, phosphorylation at Ser396 by GSK-3 or CDK5 destabilizes microtubules. Furthermore, research studies have shown that inclusions of tau are found in a number of other neurodegenerative diseases, collectively known as tauopathies (1,3).
Alternative splicing of exon 10 results in the expression of two groups of tau; three-repeat and four-repeat tau. The three-repeat tau consists of microtubule binding domains isoforms 2, 4, and 5 (Tau 3R iso2/iso4/iso5). The four-repeat tau express the tau-microtubule binding domains isoforms 6, 7, and 8 (Tau 4R iso6/iso7/iso8) (4). The localization of Tau 3R and Tau 4R in cells can be expressed differently in mild or pathological conditions, including Tau 3R in Pick's disease (PD) and corticobasal degeneration (CBD), while in Alzheimer's disease equally expresses both Tau 3R and Tau 4R (5,6). The repeat-dependent tau has a different pattern of phosphorylation in different diseases, and also has the ability and patterns of aggregation (7-9).
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