Western blot analysis of extracts from HeLa, NIH/3T3, and C6 cells using 14-3-3 τ Antibody.
Immunohistochemical analysis of paraffin-embedded human breast carcinoma, using 14-3-3 tau Antibody.
Immunohistochemical analysis of paraffin-embedded human GIST, using 14-3-3 tau Antibody.
Immunohistochemical analysis of paraffin-embedded human lung carcinoma, showing cytoplasmic and nuclear localization, using 14-3-3 tau Antibody.
Immunohistochemical analysis of paraffin-embedded human breast carcinoma, using 14-3-3 tau Antibody in the presence of control peptide (left) or antigen-specific peptide (right).
|REACTIVITY||H M R Mk|
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
NOTE: Do not allow slides to dry at any time during this procedure.
For Citrate: Heat slides in a microwave submersed in 1X citrate unmasking solution until boiling is initiated; follow with 10 min at a sub-boiling temperature (95°-98°C). Cool slides on bench top for 30 min.
|DETECTION REAGENT/SUBSTRATE COMPATIBILITY|
|SignalStain® Boost IHC Detection Reagent (HRP, Rabbit) #8114||SignalStain® Boost IHC Detection Reagent (AP, Rabbit) #18653|
|SignalStain® DAB Substrate Kit #8059||SignalStain® Vibrant Red Alkaline Phosphatase Substrate Kit #76713|
|SignalStain® Vivid Purple Peroxidase Substrate Kit #96632|
NOTE: Use of detection reagents other than those specified in this protocol may require further optimization of the primary antibody to account for the different sensitivities of the detection reagents.
posted June 2005
revised March 2016
Protocol Id: 303
14-3-3 tau Antibody detects endogenous levels of total 14-3-3 tau protein.Species Reactivity:
Human, Mouse, Rat, Monkey
Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to the sequence of human 14-3-3 tau. Antibodies are purified by protein A and peptide affinity chromatography.
The 14-3-3 family of proteins plays a key regulatory role in signal transduction, checkpoint control, apoptotic and nutrient-sensing pathways (1,2). 14-3-3 proteins are highly conserved and ubiquitously expressed. There are at least seven isoforms, β, γ, ε, σ, ζ, τ, and η that have been identified in mammals. The initially described α and δ isoforms are confirmed to be phosphorylated forms of β and ζ, respectively (3). Through their amino-terminal α helical region, 14-3-3 proteins form homo- or heterodimers that interact with a wide variety of proteins: transcription factors, metabolic enzymes, cytoskeletal proteins, kinases, phosphatases, and other signaling molecules (3,4). The interaction of 14-3-3 proteins with their targets is primarily through a phospho-Ser/Thr motif. However, binding to divergent phospho-Ser/Thr motifs, as well as phosphorylation independent interactions has been observed (4). 14-3-3 binding masks specific sequences of the target protein, and therefore, modulates target protein localization, phosphorylation state, stability, and molecular interactions (1-4). 14-3-3 proteins may also induce target protein conformational changes that modify target protein function (4,5). Distinct temporal and spatial expression patterns of 14-3-3 isoforms have been observed in development and in acute response to extracellular signals and drugs, suggesting that 14-3-3 isoforms may perform different functions despite their sequence similarities (4). Several studies suggest that 14-3-3 isoforms are differentially regulated in cancer and neurological syndromes (2,3).
Explore pathways + proteins related to this product.
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