Upstream / Downstream
Explore pathways related to this product.
CST Antibody Performance Guarantee
To Purchase # 7918S
|7918S||1 Kit (96 assays)||$489.00|
|$ 0. 00|
Find answers on our FAQs page.
PTM information and tools available.
PathScan® Total Cyclin D1 Sandwich ELISA Kit #7918
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.Learn more about how we got this image
Gallery: PathScan® Total Cyclin D1 Sandwich ELISA Kit #7918
- 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.
|Product Includes||Volume||Solution Color|
|Cyclin D1 Rabbit Antibody coated microwells||96 tests|
|Cyclin D1 Mouse Detection Antibody||11 ml||Green|
|Anti-rabbit IgG, HRP- linked Antibody||11 ml||Red|
|TMB Substrate 7004||11 ml||Colorless|
|STOP Solution 7002||11 ml||Colorless|
|Sealing Tape||2 sheets|
|ELISA Wash Buffer (20X)||25 ml||Colorless|
|ELISA Sample Diluent||25 ml||Blue|
|Cell Lysis Buffer (10X) 9803||15 ml||Yellowish|
NOTE: Refer to product-specific datasheets or product webpage for assay incubation temperature.
A. Solutions and Reagents
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
- 20X Phosphate Buffered Saline (PBS): (#9808) To prepare 1 L PBS: add 50 ml 10X PBS to 950 ml dH2O, mix.
- Bring all microwell strips to room temperature before use.
- Prepare 1X Wash Buffer by diluting 20X Wash Buffer (included in each PathScan® Sandwich ELISA Kit) in dH2O.
1X Cell Lysis Buffer: 10X Cell Lysis Buffer (#9803): To prepare 10 ml of 1X Cell Lysis Buffer, add 1 ml of 10X Cell Lysis Buffer to 9 ml of dH2O, mix. Buffer can be stored at 4°C for short-term use (1–2 weeks).
Recommended: Add 1 mM phenylmethylsulfonyl fluoride (PMSF) (#8553) immediately before use.
NOTE: Refer to product-specific datasheet or webpage for lysis buffer recommendation.
- TMB Substrate: (#7004).
- STOP Solution: (#7002).
B. Preparing Cell Lysates
For adherent cells
- Aspirate media when the culture reaches 80–90% confluence. Treat cells by adding fresh media containing regulator for desired time.
- Remove media and rinse cells once with ice-cold 1X PBS.
- Remove PBS and add 0.5 ml ice-cold 1X cell lysis buffer plus 1 mM PMSF to each plate (10 cm diameter) and incubate the plate on ice for 5 min.
- Scrape cells off the plate and transfer to an appropriate tube. Keep on ice.
- Sonicate lysates on ice.
- Microcentrifuge for 10 min (x14,000 rpm) at 4°C and transfer the supernatant to a new tube. The supernatant is the cell lysate. Store at -80°C in single-use aliquots.
For suspension cells
- Remove media by low speed centrifugation (~1,200 rpm) when the culture reaches 0.5–1.0 x 106 viable cells/ml. Treat cells by adding fresh media containing regulator for desired time.
- Collect cells by low speed centrifugation (~1,200 rpm) and wash once with 5–10 ml ice-cold 1X PBS.
- Cells harvested from 50 ml of growth media can be lysed in 2.0 ml of 1X cell lysis buffer plus 1 mM PMSF.
- Sonicate lysates on ice.
C. Test Procedure
- After the microwell strips have reached room temperature, break off the required number of microwells. Place the microwells in the strip holder. Unused microwells must be resealed in the storage bag and stored at 4°C immediately.
- Cell lysates can be undiluted or diluted with sample diluent (supplied in each PathScan® Sandwich ELISA Kit, blue color). Individual datasheets or product webpage for each kit provide information regarding an appropriate dilution factor for lysates and kit assay results.
- Add 100 µl of each undiluted or diluted cell lysate to the appropriate well. Seal with tape and press firmly onto top of microwells. Incubate the plate for 2 hr at 37°C. Alternatively, the plate can be incubated overnight at 4°C.
- Gently remove the tape and wash wells:
- Discard plate contents into a receptacle.
- Wash 4 times with 1X wash buffer, 200 µl each time per well.
- For each wash, strike plates on fresh paper towels hard enough to remove the residual solution in each well, but do not allow wells to completely dry at any time.
- Clean the underside of all wells with a lint-free tissue.
- Add 100 µl of detection antibody (green color) to each well. Seal with tape and incubate the plate at 37°C for 1 hr.
- Repeat wash procedure (Section C, Step 4).
- Add 100 µl of HRP-linked secondary antibody (red color) to each well. Seal with tape and incubate the plate for 30 min at 37°C.
- Repeat wash procedure (Section C, Step 4).
- Add 100 µl of TMB substrate to each well. Seal with tape and incubate the plate for 10 min at 37°C or 30 min at 25°C.
Add 100 µl of STOP solution to each well. Shake gently for a few seconds.
NOTE: Initial color of positive reaction is blue, which changes to yellow upon addition of STOP solution.
- Read results
- Visual Determination: Read within 30 min after adding STOP solution.
- Spectrophotometric Determination: Wipe underside of wells with a lint-free tissue. Read absorbance at 450 nm within 30 min after adding STOP solution.
posted June 2005
revised November 2013
protocol id: 21
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.
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.Species Reactivity: Human, Mouse, Rat, Monkey
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).
For Research Use Only. Not For Use In Diagnostic Procedures. Cell Signaling Technology® is a trademark of Cell Signaling Technology, Inc. PathScan® is a trademark of Cell Signaling Technology, Inc.