|Application||WB, IHC, IF,ELISA|
|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.
Specimen Preparation (forcultured cell lines, IF-IC)
1. Aspirate liquid, then cover cells to a depth of 2–3 mm with 4% formaldehyde diluted in 1X PBS.
1. Add theblocking buffer and incubate for 60 min at RT.
|Specificity||YAP1 Rabbit Recombinant mAb detects endogenous level of total YAP1.|
|Background||YAP1 is a major effector of the Hippo pathway. The YAP1/Hippo signaling pathway is a key regulator of organ size and tissue homeostasis, and its dysregulation is linked to cancer development. YAP1 activity is mediated predominantly via TEAD transcription factors. YAP1 and TEAD1 exert their transcriptional control via binding to enhancers, leading to characteristic chromatin changes and distal activation of genes. Activation of the Hippo pathway leads to phosphorylation and inactivation of the transcriptional co-activator YAP1 by cytoplasmic retention or enhanced degradation. YAP1 is phosphorylated by the Lats1 tumor suppressor, it translocates to the cytoplasm, where it interacts with 14-3-3 proteins and is thought to be inactive. YAP1 has a potent growth promoting activity and the YAP1/Hippo pathway has been tightly linked to cancer. YAP1 lacks an intrinsic DNA-binding domain and is thought to exert its co-activator function through binding to promoter sequences via interaction with transcription factors (TF), such as TEAD1/-2/-3/-4, Smads, Runx1/-2, p73, ErbB4, Pax3, AP-1, or TBX5.|