Western blot analysis of extracts from MCF7 cells, transfected with control siRNA (-) or ch-TOG siRNA (+), using Ch-TOG (D2Z8J) Rabbit mAb (upper) and α-Actinin (D6F6) XP® Rabbit mAb #6487 (lower). The Ch-TOG (D2Z8J) Rabbit mAb confirms silencing of ch-TOG expression, while the α-Actinin (D6F6) XP® Rabbit mAb is used as a loading control.
Western blot analysis of extracts from various cell lines using Ch-TOG (D2Z8J) Rabbit mAb.
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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.
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
Ch-TOG (D2Z8J) Rabbit mAb recognizes endogenous levels of total ch-TOG protein. Based on the amino acid sequence of the peptide antigen, this antibody is expected to recognize all isoforms of ch-TOG.Species Reactivity:
Human, Mouse, Rat
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Ala31 of human ch-TOG protein.
Ch-TOG (colonic hepatic tumor overexpressed gene)/CKAP5 (cytoskeleton-associated protein 5) is a microtubule stabilizing protein involved in the organization of mitotic spindle poles through interaction with the transforming acid coiled-coil protein, TACC3 (1). Ch-TOG and TACC3 also interact with the membrane trafficking protein clathrin, and this interaction is thought to be required for clathrin’s mitotic function in crosslinking microtubules in the mitotic spindle (2). Researchers have found that expression levels of both TACC3 and ch-TOG are correlated with human diseases such as glioblastoma and hepatic carcinoma (3). A genome-wide siRNA screen identified ch-TOG and other G2/M phase regulators as potential contributors to head and neck squamous cell carcinoma (4).
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