Product Pathways - PathScan ELISA
PathScan® Total PDGF Receptor α Sandwich ELISA Kit #7318
| Kit Includes | Volume | Solution Color |
|---|---|---|
| PDGF Receptor alpha Rabbit Antibody coated microwells | 96 tests | |
| PDGF Receptor alpha Mouse Detection Ab | 11 milliliters | Green |
| Anti-mouse IgG HRP-Linked Ab | 11 milliliters | Red |
| TMB Substrate | 11 milliliters | Colorless |
| STOP Solution | 11 milliliters | Colorless |
| 20X Wash Buffer | 25 milliliters | Colorless |
| Sample Diluent | 25 milliliters | Blue |
| Sealing Tape | 2 sheets | |
| Cell Lysis Buffer (10X) # 9803 | 15 milliliters | Yellowish |
Note: 12 8-well modules –Each module is designed to break apart for 8 tests.
Note: Kit should be stored at 4°C with the exception of Cell Lysis Buffer (10X), which is stored at –20°C (packaged separately).
Species Cross-Reactivity
H
Reactivity Key: H=Human
Description
CST's PathScan® Total PDGF Receptor α Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of total PDGF receptor α protein. A PDGF receptor α Rabbit Antibody* has been coated onto the microwells. After incubation with cell lysates, both phospho and nonphospho PDGF receptor α proteins are captured by the coated antibody. Following extensive washing, PDGF receptor α Mouse mAb* is added to detect both the captured phospho and nonphospho PDGF receptor α protein. Anti-mouse IgG, HRP-linked Antibody #7076* is then used to recognize the bound detection antibody. HRP substrate, TMB, is added to develop color. The magnitude of optical density for this developed color is proportional to the quantity of total PDGF receptor α protein.* Antibodies in kit are custom formulations specific to kit.
Specificity / Sensitivity
CST's PathScan® Total PDGF Receptor α Sandwich ELISA Kit #7318 detects endogenous levels of total PDGF receptor α protein. As shown in Figure 1, the level of total PDGF receptor α (phospho or nonphospho) before or after treatment of stimulator, remains unchanged as shown by Western analysis or by PathScan® Total PDGF Receptor α Sandwich ELISA Kit #7318. In Figure 3, Western blot analysis of protein captured in the PDGF Receptor α Mouse mAb coated microwell shows major bands corresponding to the PDGF receptor α protein.
Sandwich ELISA
Figure 2: The relationship between protein concentration of untreated or PDGF-treated MG63 cell lysates and the absorbance at 450 nm is shown. Cells (85% confluence) were serum starved overnight and then treated with PDGF (50 ng/ml) for 7 min. at 37?C.
Sandwich ELISA
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 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 PDGF Receptor α (D1E1E) Rabbit mAb #3174. The major bands detected in the captured material corresponds to the PDGF Receptor α protein (lanes 3 & 4).
Sandwich ELISA
Figure 1. The level of total PDGF receptor α protein (nonphospho or phospho) from either untreated or PDGF-treated MG63 cells remained similar, 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 (left panel ) or Phospho-PDGF Receptor α (Tyr754) (23B2) Rabbit mAb #2992 (right panel) is shown in the bottom figure.
Background
The proteins of the platelet derived growth factor (PDGF) family 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). PDGF Receptor α homodimers bind all PDGF isoforms except those containing PDGF D. PDGF Receptor β homodimers bind PDGF BB and DD isoforms, as well as the PDGF AB heterodimer. The heteromeric PDGFα/β receptor 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).
- Deuel, T.F. et al. (1988) Biofactors 1, 213-217.
- Bergsten, E. et al. (2001) Nat. Cell Biol. 3, 512-516.
- Betsholtz, C. et al. (2001) Bioessays 23, 494-507.
- Coughlin, S.R. et al. (1988) Prog. Clin. Biol. Res. 266, 39-45.
- Ostman, A. and Heldin, C.H. (2001) Adv. Cancer Res. 80, 1-38.
- Panayotou, G. et al. (1992) EMBO J. 11, 4261-4272.
- Ramalingam, K. et al. (1995) Bioorg. Med. Chem. 3, 1263-1272.
- Kashishian, A. et al. (1992) EMBO J. 11, 1373-1382.
Application References
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Companion Products
- 3164 PDGF Receptor α Antibody
- 2992 Phospho-PDGF Receptor α (Tyr754) (23B2) Rabbit mAb
- 9803 Cell Lysis Buffer (10X)
- 9808 Phosphate Buffered Saline (PBS-20X)
- 9809 Phosphate Buffered Saline with Tween 20 (PBST-20X)
- 7004 TMB Substrate
- 7002 STOP Solution
- 3174 PDGF Receptor α (D1E1E) Rabbit mAb
- 3170 Phospho-PDGF Receptor α (Tyr849)/PDGF Receptor β (Tyr857) (C43E9) Rabbit mAb
- 7076 Anti-mouse IgG, HRP-linked Antibody
- 7235 PathScan® Phospho-PDGF Receptor α/β (panTyr) Sandwich ELISA Kit
This product is for in vitro research use only and is not intended for use in humans or animals. This product is not intended for use as therapeutic or in diagnostic procedures.
