|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||Caspase-2 Rabbit Recombinant mAb detects endogenous level of caspase-2.|
|Background||Caspases are cysteine proteases that primarily serve two functions: the processing and activation of proinflammatory cytokines, and the cleavage of multiple proteins during apoptosis to allow the ordered dismantling of cells that are undergoing death. Among the caspases expressed in human cells, caspases 1, 4 and 5 are primarily involved in cytokine processing, whereas caspases 3, 6, 7, 8, 9 and 10 have been implicated in apoptosis. Caspases 3, 8 and 14 also have roles in cell proliferation and differentiation. Caspase 2 activation occurs during both intrinsic (mitochondrial) and extrinsic (death receptor) cell death signalling. Caspase 2 is a downstream caspase (a caspase that requires processing by an upstream initiator caspase). Caspase-2 localizes to the nucleus and has a role in the DNA damage response, cell cycle regulation and tumour suppression.|