Western blot analysis of extracts from various cell lines using CENP-T Antibody.
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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
CENP-T Antibody recognizes endogenous levels of total CENP-T protein.Species Reactivity:
Human, Mouse, Monkey
Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues near the carboxy terminus of human CENP-T protein. Antibodies are purified by protein A and peptide affinity chromatography.
Modulation of chromatin structure plays a critical role in the regulation of transcription and replication of the eukaryotic genome. The nucleosome, made up of four core histone proteins (H2A, H2B, H3, and H4), is the primary building block of chromatin. In addition to the growing number of post-translational histone modifications regulating chromatin structure, cells can also exchange canonical histones with variant histones that can directly or indirectly modulate chromatin structure (1). CENP-A, also known as the chromatin-associated protein CSE4 (capping-enzyme suppressor 4-p), is an essential histone H3 variant that replaces canonical histone H3 in centromeric heterochromatin (2). The greatest divergence between CENP-A and canonical histone H3 occurs in the amino-terminal tail of the protein, which binds linker DNA between nucleosomes and facilitates proper folding of centromeric heterochromatin (3). The amino-terminal tail of CENP-A is also required for recruitment of other centromeric proteins (CENP-C, hSMC1, hZW10), proper kinetochore assembly, and chromosome segregation during mitosis (4).
CENP-A is regarded as the epigenetic mark of the centromere that persists through cell generations (5). Although its presence is necessary, it is not sufficient for formation of functional kinetochores (6). CENP-T, in complex with CENP-W, has recently been shown to form a histone fold, a structure that is capable of association with DNA, and target DNA to the kinetochore (7). Kinetochore attachment is mediated by a long flexible N-terminal region that has been shown to interact with outer proteins of the kinetochore complex (reviewed in 8). Moreover, the CENP-T-W complex has also been shown to interact with the CENP-S-X dimer, to form a heterotetrameric complex that has structural and potentially functional similarity to canonical histones (8). Since CENP-S-X are conserved kinetochore localized proteins, this new complex has been suggested to be a novel centromeric histone.
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