Figure 1: Constitutive phosphorylation of YAP at Ser127 in PANC-1 cells is detected by PathScan® Phospho-YAP (Ser127) Sandwich ELISA Kit. In contrast, a low level of phospho-YAP at Ser127 protein is detected in λ-phosphatase-treated PANC-1 cells (nonphospho lysate). The absorbance readings at 450 nm are shown in the top figure, while the corresponding Western blots using Phospho-YAP (Ser127) (D9W2I) Rabbit mAb #13008 or YAP Antibody #4912 are shown in the bottom figure.
Figure 2: The relationship between protein concentration of lysates from untreated and λ-phosphatase-treated PANC-1 cells and the absorbance at 450 nm is shown. Unstarved PANC-1 cells (85-90% confluence) were treated with λ-phosphatase, or left untreated, and then lysed with Cell Lysis Buffer (#9803).
|Product Includes||Volume||Solution Color|
|YAP Rabbit mAb Coated Microwells||96 tests|
|Phospho-YAP (Ser127) Rabbit Detection mAb (Biotinylated)||1 ea||Green (Lyophilized)|
|HRP-Linked Streptavidin (ELISA Formulated)||1 ea||Red (Lyophilized)|
|Detection Antibody Diluent||11 ml||Green|
|HRP Diluent||11 ml||Red|
|TMB Substrate 7004||11 ml|
|STOP Solution 7002||11 ml|
|Sealing Tape||2 ea|
|ELISA Wash Buffer (20X) 9801||25 ml|
|ELISA Sample Diluent||25 ml||Blue|
|Cell Lysis Buffer (10X) 9803||15 ml|
The PathScan® Phospho-YAP (Ser127) Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of YAP protein phosphorylated at Ser127. A YAP rabbit antibody has been coated onto the microwells. After incubation with cell lysates, both phospho- and non-phospho-YAP proteins are captured by the coated antibody. Following extensive washing, a phospho-YAP (Ser127) biotinylated mAb is added to detect the captured phospho-YAP protein. An HRP-linked Streptavidin is then used to recognize the bound detection antibody. HRP substrate, TMB, is added to develop color. The magnitude of the absorbance for this developed color is proportional to the quantity of YAP phosphorylated at Ser127.
Antibodies in this kit are custom formulations specific to kit.
NOTE: Prepare solutions with purified water.
*NOTE: Some PathScan® ELISA Kits may include HRP-Linked Streptavidin in place of HRP-Linked Antibody.
NOTE: Initial color of positive reaction is blue, which changes to yellow upon addition of STOP Solution.
posted November 2013
Protocol Id: 204
PathScan® Phospho-YAP (Ser127) Sandwich ELISA Kit detects endogenous levels of YAP protein phoshorylated at Ser127 in human cells, as shown in Figure 1. The kit sensitivity is shown in Figure 2. This kit detects proteins from the indicated species, as determined through in-house testing, but may also detect homologous proteins from other species.
YAP (Yes-associated protein, YAP65) was first identified based on its ability to associate with the SH3 domain of Yes. It also binds to other SH3 domain-containing proteins such as Nck, Crk, Src, and Abl (1). In addition to the SH3 binding motif, YAP contains a PDZ interaction motif, a coiled-coil domain, and WW domains (2-4). While initial studies of YAP all pointed towards a role in anchoring and targeting to specific subcellular compartments, subsequent studies showed that YAP is a transcriptional co-activator by virtue of its WW domain interacting with the PY motif (PPxY) of the transcription factor PEBP2 and other transcription factors (5). In its capacity as a transcriptional co-activator, YAP is now widely recognized as a central mediator of the Hippo Pathway, which plays a fundamental and widely conserved role in regulating tissue growth and organ size (6-8). Phosphorylation at multiple sites (e.g., Ser109, Ser127) by LATS kinases promotes YAP translocation from the nucleus to the cytoplasm, where it is sequestered through association with 14-3-3 proteins (7-9). These LATS-driven phosphorylation events serve to prime YAP for subsequent phosphorylation by CK1δ/ε in an adjacent phosphodegron, triggering proteasomal degradation of YAP (10).
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