Western blot analysis of extracts from HeLa cells, untreated (-) or treated with Trichostatin A (TSA) #9950 (400 nM, 16 hr; +), using Acetyl-α-Tubulin (Lys40) (D20G3) XP® Rabbit mAb (HRP Conjugate).
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This Cell Signaling Technology antibody is conjugated to the carbohydrate groups of horseradish peroxidase (HRP) via its amine groups. The HRP conjugated antibody is expected to exhibit the same species cross-reactivity as the unconjugated Acetyl-α-Tubulin (Lys40) (D20G3) XP® Rabbit mAb #5335.
Supplied in 136 mM NaCl, 2.6 mM KCl, 12 mM sodium phosphate (pH 7.4) dibasic, 2 mg/ml BSA, and 50% glycerol. Store at –20°C. Do not aliquot the antibodies.
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.
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 June 2016
Protocol Id: 264
Acetyl-α-Tubulin (Lys40) (D20G3) XP® Rabbit mAb (HRP Conjugate) detects endogenous levels of α-tubulin only when acetylated at Lys40. This amino acid is not conserved in β-tubulin.Species Reactivity:
Human, Mouse, Rat, Monkey, ZebrafishSpecies predicted to react based on 100% sequence homology:
Monoclonal antibody is produced by immunizing animals with a synthetic acetylpeptide corresponding to residues surrounding Lys40 of human α-tubulin protein.
The cytoskeleton consists of three types of cytosolic fibers: microtubules, microfilaments (actin filaments), and intermediate filaments. Globular tubulin subunits comprise the microtubule building block, with α/β-tubulin heterodimers forming the tubulin subunit common to all eukaryotic cells. γ-tubulin is required to nucleate polymerization of tubulin subunits to form microtubule polymers. Many cell movements are mediated by microtubule action, including the beating of cilia and flagella, cytoplasmic transport of membrane vesicles, chromosome alignment during meiosis/mitosis, and nerve-cell axon migration. These movements result from competitive microtubule polymerization and depolymerization or through the actions of microtubule motor proteins (1).
The Elongator complex catalytic subunit (Elp3) acetylates α-tubulin at Lys40 while the histone deacetylase HDAC6 functions as a tubulin deacetylase. This post-transcriptional modification may be required for dynamic cell shape remodeling, cell motility, tubulin stability and terminal branching of cortical neurons (2,3).
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