Western blot analysis of extracts from 293 cells, mock transfected (-) or transfected with a construct expressing Myc/DDK-tagged full-length human NKX2.2 (hNKX2.2-Myc/DDK; +), using NKX2.2 Antibody (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower).
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
NKX2.2 Antibody recognizes transfected levels of total NKX2.2 protein.Species Reactivity:
HumanSpecies predicted to react based on 100% sequence homology:
Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Ala92 of human NKX2.2 protein. Antibodies are purified by protein A and peptide affinity chromatography.
The NKX family of homeobox genes are known to act as intermediaries in the neural response to Sonic hedgehog signaling during central nervous system development (1). NKX2.2 is a member of this family of transcription factors and is necessary for neuroendocrine differentiation in the central nervous system and pancreas (2,3). NKX2.2 mutant mice die shortly after birth due to incomplete differentiation of insulin-producing pancreatic β cells and defects in ventral neural patterning (2,3). According to the research literature, expression of NKX2.2 has also been found in neuroendocrine tumors of the gut, making it a potential marker for the study of gastrointestinal neuroendocrine tumors (4).
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