Western blot analysis of extracts from HT-29 and HeLa cells, untreated (-) or treated with Nocodazole #2190 (100 ng/mL, 24 hr; +), using CENP-E (E5N7B) Rabbit mAb (upper) or α-Actinin (D6F6) XP® Rabbit mAb #6487 (lower).Learn more about how we get our images.
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
Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide. Store at –20°C. Do not aliquot the antibody.
CENP-E (E5N7B) Rabbit mAb recognizes endogenous levels of total CENP-E protein.
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Pro333 of human CENP-E protein.
Centromere-associated protein E (CENP-E) is a kinesin-like motor protein and mitotic-checkpoint kinase BUB1B binding partner that is essential for establishing and maintaining stable attachments between mitotic chromosomes and spindle microtubules (1). CENP-E plays an important role as a motor protein in the alignment of chromosomes during prometaphase (2). Research studies indicate that CENP-E protein expression peaks in late G2 and M-phases of the cell cycle before the protein is degraded at mitotic exit (3). Additional studies show that the loss of CENP-E function results in cell cycle arrest in mitosis. Mutations in the corresponding CENPE gene can result in autosomal recessive primary microcephaly-13, a developmental disorder characterized by small head circumference, dysmorphic facial features, short stature, and delayed psychomotor development (4). Since CENP-E is essential for mitotic progression and is required for cellular proliferation, it has become an interesting target for cancer therapy (5-7).
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc. XP is a registered trademark of Cell Signaling Technology, Inc. Tween is a registered trademark of ICI Americas, Inc.
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