|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.
NOTE: Do not allow slides to dry at any time during this procedure.
1. Deparaffinize/hydrate sections:
1. Incubate sections in three washes of xylene for 5 min each.
2. Wash sections two times in dH2O for 5 min each.
For Citrate: Heat slides in a microwave submersed in 1X citrate unmasking solution until boiling is initiated; continue with 10 min at a sub-boiling temperature (95°-98°C). Cool slides on bench top for 30 min.
1. Wash sections in dH2O three times for 5 min each.
|Specificity||EBP50 Rabbit Recombinant mAb detects endogenous level of total EBP50.|
|Background||EBP50 (also known as NHERF1), a 55-kDa phosphoprotein, was first identified as a cofactor essential for protein kinase A-mediated inhibition of Na+/H+ exchanger isoform 3 (NHE3). EBP50 contains two PDZ domains (PDZ1 and PDZ2) implicated in multiple protein-protein interactions, and an ERM domain, which binds to the actin-associated ERM proteins (ezrin, radixin, moesin, and merlin). EBP50 was also found to interact with a small number of transmembrane proteins such as the cystic fibrosis transmembrane conductance regulator (CFTR), the P2Y1 purinergic receptor, the platelet-derived growth factor receptor, the β2-adrenergic receptor, the B1 subunit of the H+-ATPase, and the type IIa sodium phosphate cotransporter. EBP50 also associates with the phospholipases C (PLC)-β1/β2, and with the TRP4 and TRP5 calcium channels to form a PLC-β1/2-TRP4/5-EBP50 protein complex. The EBP50 protein can also bind through its PDZ domains to various intracellular proteins, including GRK6A, EPI64, and Yes-associated protein 65.|