Figure 1. Cyclin D1 protein from human (HT-1080 and MCF7), monkey (COS-7), mouse (C2C12), and rat (C6) cells was detected using PathScan® Total Cyclin D1 Sandwich ELISA Kit #7918. However, this kit has a low detection level for cyclin D1 in some cell lines, such as HeLa, Mv 1 Lu or NIH/3T3. The absorbance readings at 450 nm are shown in the top figure, while the corresponding western blot using Cyclin D1 Antibody #2922, is shown in the bottom figure.
Figure 2. The relationship between protein concentration of lysates from HT-1080 cells and the absorbance at 450 nm is shown. HT-1080 cells (80% confluence) were harvested and then lysed.
|REACTIVITY||H Mk M R|
|Product Includes||Volume||Solution Color|
|Cyclin D1 Rabbit Ab Coated Microwells||96 tests|
|Cyclin D1 Mouse Detection mAb||1 ea||Green (Lyophilized)|
|Anti-mouse IgG, HRP-linked Antibody (ELISA Formulated)||1 ea||Red|
|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® Total Cyclin D1 Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of total cyclin D1 protein. A Cyclin D1 Rabbit Antibody has been coated onto the microwells. After incubation with cell lysates, both phospho and nonphospho cyclin D1 proteins are captured by the coated antibody. Following extensive washing, Cyclin D1 Mouse Detection Antibody is added to detect the captured cyclin D1 protein. Anti-mouse IgG, HRP-linked Antibody is then used to recognize the bound detection antibody. HRP substrate, TMB, is added to develop color. The magnitude of absorbance for this developed color is proportional to the quantity of total cyclin D1 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® Total Cyclin D1 Sandwich ELISA Kit detects endogenous levels of cyclin D1 protein in human, monkey, mouse, and rat 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.
Human, Monkey, Mouse, Rat
Activity of the cyclin-dependent kinases CDK4 and CDK6 is regulated by T-loop phosphorylation, by the abundance of their cyclin partners (the D-type cyclins), and by association with CDK inhibitors of the Cip/Kip or INK family of proteins (1). The inactive ternary complex of cyclin D/CDK4 and p27 Kip1 requires extracellular mitogenic stimuli for the release and degradation of p27 concomitant with a rise in cyclin D levels to affect progression through the restriction point and Rb-dependent entry into S-phase (2). The active complex of cyclin D/CDK4 targets the retinoblastoma protein for phosphorylation, allowing the release of E2F transcription factors that activate G1/S-phase gene expression (3). Levels of cyclin D protein drop upon withdrawal of growth factors through downregulation of protein expression and phosphorylation-dependent degradation (4).
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