Figure 1. Treatment of MG63 cells with PDGF stimulates tyrosine phosphorylation of PDGF Receptor α/β, detected by PathScan® Phospho-PDGF Receptor α/β (panTyr) Sandwich ELISA Kit #7235, but does not affect the level of total PDGF Receptor α or β protein detected by either PathScan® Total PDGF Receptor α Sandwich ELISA Kit #7318 or Western analysis. The absorbance readings at 450 nm are shown in the top figure, while the corresponding Western blot using PDGF Receptor α Rabbit mAb #3174 (panel A), PDGF Receptor β Rabbit mAb #3169 (panel B), Phospho-PDGF Receptor α (Tyr754) (23B2) Rabbit mAb #2992 (panel C) or Phospho-PDGF Receptor β (Tyr771) (76D6) Rabbit mAb #3173 (panel D) is shown in the bottom figure.
Figure 2. The relationship between protein concentration of untreated or PDGF-treated MG63 cell lysates and the absorbance at 450 nm is shown. Cells were serum starved overnight and then treated with PDGF (50 ng/ml) for 7 min. at 37oC.
Figure 3. Kit specificity as demonstrated by Western analysis of the ELISA microwell captured protein. Lysates were prepared from MG63 cells and incubated in microwells coated with the PDGF Receptor α/β capture antibody. Wells were washed, and the captured protein was solubilized in SDS gel loading buffer. Western analysis of MG63 cell starting lysate (lanes 1 & 2) and the captured protein (lanes 3 & 4) was performed using Phospho-Tyrosine Mouse mAb (P-Tyr-100) (Biotinylated) #9417.
The major band detected in the captured material corresponds to the phospho-PDGF Receptor α/β protein (lanes 3 & 4).
|Product Includes||Volume||Solution Color|
|PDGFR alpha/beta Rabbit mAb Coated Microwells||96 tests|
|Phospho-Tyrosine Mouse 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-PDGF Receptor α/β (panTyr) Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of PDGF receptor α/β when tyrosine phosphorylated. A PDGF Receptor α/β Rabbit Antibody has been coated onto the microwells. After incubation with cell lysates, PDGF Receptor α/β (phospho and nonphospho) is captured by the coated antibody. Following extensive washing, a Biotinylated Phospho-Tyrosine Mouse Detection Antibody is added to detect tyrosine phosphorylation of the captured PDGF receptor α/β protein. HRP-linked Strepavidin 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 PDGF receptor α/β phosphorylated on tyrosine.
Antibodies in 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
CST's PathScan® Phospho-PDGF Receptor α/β (panTyr) Sandwich ELISA Kit #7235 detects PDGF receptor α/β when tyrosine phosphorylated. As shown in Figure 1, a significant induction of PDGF Receptor α/β tyrosine phosphorylation can be detected in MG63 cells following treatment with PDGF using the Phospho-PDGF Receptor α/β (panTyr) Sandwich ELISA Kit #7235. The level of total PDGF receptor α (phospho and nonphospho) remains unchanged as shown by Western analysis and by PathScan® Total PDGF Receptor α Sandwich ELISA Kit #7318. Western blot analysis of protein captured in the PDGF receptor α/β antibody coated microwell indicates that PDGF receptor α/β (phospho and nonphospho) has been captured (data not shown). The Western blot also shows a major band corresponding to the phospho-PDGF receptor α/β protein when biotinylated anti-tyrosine antibody is used as probe (see Figure 3). This kit detects proteins from the indicated species, as determined through in-house testing, but may also detect homologous proteins from other species.Species Reactivity:
Platelet derived growth factor (PDGF) family proteins exist as several disulphide-bonded, dimeric isoforms (PDGF AA, PDGF AB, PDGF BB, PDGF CC, and PDGF DD) that bind in a specific pattern to two closely related receptor tyrosine kinases, PDGF receptor α (PDGFRα) and PDGF receptor β (PDGFRβ). PDGFRα and PDGFRβ share 75% to 85% sequence homology between their two intracellular kinase domains, while the kinase insert and carboxy-terminal tail regions display a lower level (27% to 28%) of homology (1). PDGFRα homodimers bind all PDGF isoforms except those containing PDGF D. PDGFRβ homodimers bind PDGF BB and DD isoforms, as well as the PDGF AB heterodimer. The heteromeric PDGF receptor α/β binds PDGF B, C, and D homodimers, as well as the PDGF AB heterodimer (2). PDGFRα and PDGFRβ can each form heterodimers with EGFR, which is also activated by PDGF (3). Various cells differ in the total number of receptors present and in the receptor subunit composition, which may account for responsive differences among cell types to PDGF binding (4). Ligand binding induces receptor dimerization and autophosphorylation, followed by binding and activation of cytoplasmic SH2 domain-containing signal transduction molecules, such as GRB2, Src, GAP, PI3 kinase, PLCγ, and NCK. A number of different signaling pathways are initiated by activated PDGF receptors and lead to control of cell growth, actin reorganization, migration, and differentiation (5). Tyr751 in the kinase-insert region of PDGFRβ is the docking site for PI3 kinase (6). Phosphorylated pentapeptides derived from Tyr751 of PDGFRβ (pTyr751-Val-Pro-Met-Leu) inhibit the association of the carboxy-terminal SH2 domain of the p85 subunit of PI3 kinase with PDGFRβ (7). Tyr740 is also required for PDGFRβ-mediated PI3 kinase activation (8).
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