|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.
Cyclin D1 Rabbit Recombinant mAb detects endogenous levels of cyclin D1.
Cyclin D1 serves as a key sensor and integrator of extracellular signals of cells in early to mid-G1 phase, mediating its function through binding both the CDKs and histone acetylase [p300/cAMP response element-binding protein-binding protein (CBP) and P/CAF] and histone deacetylases to modulate local chromatin structure of the genes that are involved in regulation of cell proliferation and differentiation. Cyclin D1 is the regulatory subunit of the holoenzymes that phosphorylate and, together with sequential phosphorylation by cyclin E/CDK2, inactivate the cell-cycle inhibiting function of the retinoblastoma protein (pRb). pRb serves as a gatekeeper of the G1 phase, and passage through the restriction point leads to DNA synthesis. Cyclin D1 is also essential in cellular adhesion, motility, and guided migration of primary bone marrow macrophages. Cyclin D1 physically associates with transcriptional factors or coactivators including HATs and HDACs to regulate transcription and epigenetic changes.