|Reactivity||Human Mouse Rat|
|Storage buffer||10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide.|
|Storage||Store at –20°C.|
1. Aspirate media from cultures and Wash the cells with 1X PBS. 2. Lyse cells by adding 1X SDS sample buffer and transfer the extract to a microcentrifuge tube. Keep onice. 3. Sonicate for 10–15 sec to complete cell lysis and shear DNA. 4. Heat a 20 µl sample to 95–100°C for 5 min, then cool on ice. 5. Centrifuge for 5 min (with Microcentrifuge). 6. Load appropriate volumes of samples onto SDS-PAGE gel (loading quantity of protein sample depends on the concentration of extracted proteins). NOTE: At the same time, please load the pre-stained molecular weight markers to determine molecular weights and verify electrotransfer. 7. Electrotransfer to nitrocellulose/PVDF membrane.
Membrane Blocking and Antibody Incubations
1. (Optional) After transfer, wash the transferred membrane with TBS for 5 min at room temperature. 2. Incubate the membrane in the blocking buffer for 1 hr at room temperature. 3. Wash three times for 5 min each with TBST.
b. Antibodies Incubation
1. Incubate membrane and primary antibody (at the appropriate dilution and diluent recommended) in a primary antibody dilution buffer with gentle agitation overnight at 4°C. 2. Wash three times for 5 min each with TBST. 3. Incubate membrane with an appropriate second antibodydissolved in the blocking buffer with gentle agitation for 1 hr at room temperature. 4. Wash three times for 5 min each with TBST. 5. Proceed with detection.
Detection of Proteins
1. After antibodies incubation, Wash membrane three times for 5 minutes in TBST. 2. PrepareECL Reagent (or other chromogenic agents/substrate according to your second antibody). Mix well. 3. Incubate substrate with membrane for 1 minute, remove excess solution (membrane remains wet), wrap in plastic and expose to X-ray film.
|Specificity||LSD2/AOF1 Rabbit Recombinant mAb detects endogenous level of total LSD2/AOF1.|
|Background||Histone lysine demethylases (KDMs) are family of enzymes which are involved in the extensive epigenetic modification in many cellular processes such as transcription regulation, chromatin remodeling, DNA proofreading and repair, cellular proliferation, embryotic development, etc. To date, two families of histone demethylases (KDMs) have been discovered; the flavin-dependent lysine specific demethylases and the JmjC-domain containing KDMs. The flavin-dependent KDM family includes LSD1 (KDM1A/AOF2) and LSD2 (KDM1B/AOF1). Structurally, LSD1 contains a coiled-coil tower domain protruding from the AO domain responsible for interaction with its co-factors, while LSD2 possesses an aminoterminal zinc finger element that is necessary for LSD2 binding to its methylated substrate. Although LSD1 and LSD2 share significant homology of amino acid sequence in the AO domain and both enzymes demethylate lysine 4 on histone 3 in a FAD-dependent manner, it is apparent that the two enzymes also have distinct functions, and therefore may act differently in regulation of gene transcription and chromatin remodeling. While LSD1 mostly binds to the promoter region of genes, LSD2 associates primarily with the body regions of actively transcribed genes. LSD2 is not only a histone demethylase but also functions as an E3 ubiquitin ligase.|