Figure 1: Treatment of serum-starved WI-38 cells (3 days) with serum for 2 days induces phospho-Rb (Ser780), as detected by the PathScan® Phospho-Rb (Ser780) Sandwich ELISA Kit #13016. The absorbance readings at 450 nm are shown in the top figure while the corresponding western blot using Phospho-Rb (Ser780) (D59B7) Rabbit mAb #8180 is shown in the bottom figure.
Figure 2: The relationship between protein concentration of lysates from Jurkat (Rb-positive) and Saos-2 (Rb-negative) cells and the absorbance at 450 nm is shown, as detected by the PathScan® Phospho-Rb (Ser780) Sandwich ELISA Kit #13016.
|Product Includes||Volume||Solution Color|
|Phospho-Rb (Ser780) Rabbit mAb Coated Microwells||96 tests|
|Rb Mouse Detection mAb||1 ea||Green (Lyophilized)|
|Anti-mouse IgG, HRP-linked Antibody (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|
PathScan® Phospho-Rb (Ser780) Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of phospho-Rb (Ser780) protein. A Phospho-Rb (Ser780) specific antibody has been coated onto the microwells. After incubation with cell lysates, phospho-Rb (Ser780) protein is captured by the coated antibody. Following extensive washing, a Rb mouse mAb is added to detect the captured phospho-Rb protein. HRP-linked Anti-Mouse IgG 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 phospho-Rb (Ser780) protein.
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
PathScan® Phospho-Rb (Ser780) Sandwich ELISA Kit detects endogenous levels of Rb protein when phosphorylated at Ser780, as shown in figure 1. The kit sensitivity is shown in Figure 2. Phosphatase treatment, as well as dot blot analysis (data not shown) using specific phosphopeptides and non-phosphopeptides, was used to confirm the phospho-specificity of the antibodies provided in this kit. This kit detects proteins from the indicated species, as determined through in-house testing, but may also detect homologous proteins from other species.
The retinoblastoma tumor suppressor protein Rb regulates cell proliferation by controlling progression through the restriction point within the G1-phase of the cell cycle (1). Rb has three functionally distinct binding domains and interacts with critical regulatory proteins including the E2F family of transcription factors, c-Abl tyrosine kinase, and proteins with a conserved LXCXE motif (2-4). Cell cycle-dependent phosphorylation by a CDK inhibits Rb target binding and allows cell cycle progression (5). Rb inactivation and subsequent cell cycle progression likely requires an initial phosphorylation by cyclin D-CDK4/6 followed by cyclin E-CDK2 phosphorylation (6). Specificity of different CDK/cyclin complexes has been observed in vitro (6-8) and cyclin D1 is required for Ser780 phosphorylation in vivo (9).
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