|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||Phospho-RPA2 (T21) Rabbit Recombinant mAb detects endogenous level of total phospho-RPA2 (T21).|
|Background||One factor that plays essential roles both during DNA replication and in the repair- and recombination-mediated recovery from damage is replication protein A (RPA), the eukaryotic single-stranded (ss) DNA-binding protein. RPA is a heterotrimeric protein consisting, in mammalian cells, of ∼70- (RPA1), 30- (RPA2), and 14 (RPA3)-kDa subunits. During DNA replication, RPA acts at the fork, stabilizing ssDNA and facilitating nascent strand synthesis by the replicative DNA polymerases. Under DNA-damaging conditions, RPA-ssDNA complexes act to recruit and activate a key checkpoint mediator consisting of the ATR and ATRIP (ATR-interacting protein) protein-kinase complex. At DNA damage-dependent nuclear foci, RPA interacts with repair and recombination components to process double-strand DNA breaks and other lesions. RPA activity is regulated by various stress conditions. In particular, heat shock, exposure to UV radiation, and treatment with DNA-alkylating agents each cause the generation of an RPA species that is unable to support DNA replication in vitro. Two of the RPA2 sites (S23 and S29) are phosphorylated in a cell cycle-dependent manner by cyclin-cdk2 complexes. At least five of the other seven (S4, S8, S11, S12, S13, T21, and S33) can be phosphorylated in response to UV irradiation.|