Western blot analysis of extracts from various cell lines using Mis18-α (D3N2X) Rabbit mAb.
Western blot analysis of extracts from 293T cells, mock transfected (-) or transfected (+) with full length human Mis18-α/Myc-DDK, using Mis18-α (D3N2X) Rabbit mAb (upper) or Myc-Tag (71D10) Rabbit mAb #2278 (lower).
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
Mis18-α (D3N2X) Rabbit mAb recognizes endogenous levels of total Mis18-α protein.Species Reactivity:
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Arg41 of human Mis18-α protein.
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. The inherited localization of the centromere is specified by CENP-A (1). CENP-A deposition to the correct chromosomal location in early G1 phase is regulated by the Mis18 complex, which consists of Mis18-alpha, Mis18-beta, Mis18BP1, RbAp48 and RbAp46 (2).
Mis18-alpha deficiency in mice results in inappropriate localization of CENP-A, as well as DNA methylation defects (3). Localization of the Mis18 complex to centromeres is regulated by the mitotic kinase Plk1 (polo-like kinase 1) (4).
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