|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||HDAC7 Rabbit Recombinant mAb detects endogenous level of total HDAC7.|
|Background||HDAC7 is a class II histone deacetylase. The class IIa HDACs play important roles in the developmentally regulated expression of genes involved in the differentiation and function of muscle, immune, and neural cells. HDACs catalyze the removal of acetyl groups from lysine residues in the N-terminal regions of the core histone octamer subunits. The class IIa HDACs, comprising the subfamily members HDAC4, -5, -7, and -9, are defined by the presence of a large N-terminal domain that mediates both recruitment to specific promoters and signal-dependent shuttling between the nucleus and the cytoplasm. HDAC4, HDAC5, and HDAC7 function as transcriptional corepressors recruited by the MEF2 transcription factors and the N-CoR, BCoR, and CtBP corepressors. They are regulated through subcellular compartmentalization controlled by site-specific phosphorylation and binding of 14-3-3 proteins.|