|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.
Cleaved PARP Rabbit Recombinant mAb detects endogenous levels of cleaved PARP.
Apoptosis plays important roles in development, immunological competence, and homeostasis. It is characterized by marked changes in cellular morphology, including chromatin condensation, membrane blebbing, nuclear breakdown, and the appearance of membrane-associated apoptotic bodies, internucleosomal DNA fragmentation, as well as by cleavage of poly(ADP-ribose) polymerase (PARP). PARP catalyzes the poly(ADP-ribosyl)ation of a variety of nuclear proteins with NAD as substrate. Because it is activated by binding to DNA ends or strand breaks, PARP was suggested to contribute to cell death by depleting the cell of NAD and ATP. PARP was subsequently shown to be cleaved into 89- and 24-kDa fragments that contain the active site and the DNA-binding domain of the enzyme, respectively, during drug-induced apoptosis in a variety of cells. Such cleavage essentially inactivates the enzyme by destroying its ability to respond to DNA strand breaks. caspase 3 is primarily responsible for the cleavage of PARP during cell death. Cleavage of PARP-1 by caspase-3 has been implicated in several neurological diseases e.g. cerebral ischemia, Alzheimer's disease, multiple sclerosis, Parkinson's disease, traumatic brain injury, NMDA-mediated excitotoxicity and brain tumors, especially gliomas. Besides caspase-3, caspase-7 also cleaves PARP-1 in vivo. Almost all caspases including caspase-1, are known to modify PARP-1 in vitro.