Western blot analysis of extracts from HCT 116, PC-12, and COS-7 cell lines using LSD2 (E1R6O) Rabbit mAb.
Western blot analysis of extracts from wild-type (WT), LSD2 knockout (LSD2 KO), and LSD1 knockout (LSD1 KO) HeLa cells using LSD2 (E1R6O) Rabbit mAb (upper) and α-Actinin (D6F6) XP® Rabbit mAb #6487 (lower). As expected, LSD2 is still expressed in LSD1 knockout HeLa cells.
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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
LSD2 (E1R6O) Rabbit mAb recognizes endogenous levels of total LSD2 protein. This antibody does not cross-react with LSD1 protein.Species Reactivity:
Human, Mouse, Rat, Monkey
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Leu467 of human LSD2 protein.
Lysine-specific demethylase 2 (LSD2; also known as AOF1) is a nuclear amine oxidase homolog that acts as a histone demethylase and transcription cofactor protein (1,2). LSD2 functions as a co-repressor protein by demethylating mono-methyl and di-methyl histone H3 Lys4, two marks associated with actively transcribed genes (1,2). LSD2-mediated demethylation of histone H3 Lys4 is required for establishing proper DNA methylation imprints during oogenesis (3). In addition, LSD2 appears to be overexpressed in malignant breast cancers, where it contributes to DNA methylation and repression of multiple tumor suppressor genes (4,5). Furthermore, LSD2 also contains E3 ubiquitin ligase activity that targets O-GlcNac transferase (OGT) for proteosomal degradation (6). A549 lung cancer cell growth is dependent on this E3 ubiquitin ligase activity, suggesting that this function of LSD2 is also important for proper gene regulation (6).
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