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Pathway Focus: Cell Cycle
We now offer ELISA kits for measuring p27Kip1 and p21 Waf1/Cip1 protein levels in cell lysates. Both proteins are members of cyclin-dependent kinase (CDK) inhibitors and are negative cell cycle regulators that play an important role in tumor suppression. Cell division relies on the activation of cyclins, which bind to CDKs to induce cell cycle progression toward S phase and later to initiate mitosis. Uncontrolled CDK activity is often the cause of human cancer; CDK function is tightly regulated by cell-cycle inhibitors such as p21 Waf1/Cip1 and p27Kip1 proteins. Following anti-mitogenic signals or DNA damage, p21 Waf1/Cip1 and p27Kip1 bind to cyclin–CDK complexes to inhibit their catalytic activity and induce cell cycle arrest.
|ELISA kits provide a simple and unbiased way to quantify specific proteins. ELISA kits provide better sensitivity than western blots, allowing faint bands to be detected with greater certainty, and less sample lysate is needed for results. In addition, results are quantitative and done in the same day.|
Antibodies are coated on the bottom of a 96-well plate (immobilized). Sample is added to the well, and the immobilized capture antibody binds to the protein antigen. Nonspecific binding is washed away, and then a detector antibody is added. A secondary antibody conjugated to the enzyme horseradish peroxidase (HRP) is added. A chromogen substrate is added to react with any bound enzyme to produce a color, which is directly proportional to the concentration of the protein present. A stop solution is then added to terminate the color reaction, and the intensity is measured using a plate reader.
|Figure 1. Natural p27Kip1 from serum-starved MCF 7 cell lysate was serially diluted in standard diluent buffer. The parallelism shown between the standard and MCF-7 lysate indicates that the standard accurately reflects p27Kip1 content in samples.|
|Figure 2. Apicidin treatment (2 µg/mL for 24 hr) increases p21 Waf1/Cip1 expression level in MCF 7 cells. Untreated MCF 7 cells were used as a control. Cell extracts were prepared and cell lysates (200 µg/mL) were analyzed with the p21 Waf1/Cip1 ELISA Kit.|
|p21 Waf1/Cip1 ELISA Kit||96 tests||KHO5421|
|p27Kip1 ELISA Kit||96 tests||KHO5321|
Premo™ FUCCI Cell Cycle Sensor is a fluorescent protein–based system based on the Fluorescence Ubiquitination Cell Cycle Indicator (FUCCI) developed by Miyawaki and colleagues that employs a red fluorescent protein (RFP) and a green fluorescent protein (GFP), each fused to a different cell cycle regulator: Cdt1 or geminin. During the cell cycle, these two proteins are ubiquitinated by specific ubiquitin E3 ligases, which target them to the proteasome for degradation. E3 ligases display temporal regulation of activity, resulting in biphasic cycling of geminin and Cdt1 levels during the cell cycle. A dynamic color change (red to yellow to green) clearly displays progression through the cell cycle and division. Premo™ FUCCI Cell Cycle Sensor can be used not only to identify which phase of the cell cycle the cell is in, but also to track the time spent in each phase of the cell cycle.
|We have combined FUCCI with the powerful BacMam gene delivery system to form the Premo™ FUCCI Cell Cycle Sensor. Baculoviruses have typically been used over the last several decades for high-level protein production in insect cells. Recently, modified baculovirus containing a mammalian promoter, known as BacMam virus, has been shown to give high transduction efficiencies across several cell types with minimal cytopathic effects. BacMam does not replicate in mammalian cells, thereby offering a transient footprint-free method to label cells for downstream applications. The genetically encoded and prepackaged reagents are ready for immediate use—there’s no need to purify plasmid or worry about vector integrity and quality. |
Transduction is efficient and reproducible in most cell types, including primary and stem cells, without apparent cytopathic effects. No lipids, dye-loading chemicals, or other potentially harmful treatments are required. In addition, BacMam technology allows you to precisely titrate expression levels to optimize the reagent for your experiment.
|Figure 3. Live-cell imaging with Premo™ FUCCI Cell Cycle Sensor. U2OS cells were transduced with Premo™ FUCCI Cell Cycle Sensor and costained with the far red–fluorescent Alexa Fluor® 647 wheat germ agglutinin. Imaging was performed on live cells using a Delta Vision Core microscope and standard FITC/TRITC/Cy®5 filter sets.|
|Figure 4. Two-color cell cycle mapping using Premo™ FUCCI Cell Cycle Sensor.|
|Premo™ FUCCI Cell Cycle Sensor||1 kit||P36232|
Proliferation is traditionally assessed by incubating cells with a single “pulse” of a nucleoside analog (e.g., bromodeoxyuridine, BrdU) that is incorporated into DNA and detected with antibodies. Dual pulse labeling typically relies on BrdU immunocytochemistry with iododeoxyuridine (IdU) or chlorodeoxyuridine (CldU), using multiple BrdU antibodies of different clones that cross-react with IdU and CldU for detection, but also often react with BrdU.
|However, by using sequential pulses of the thymidine analogs EdU and BrdU, this complex method is greatly simplified. Rather than relying on antibody-based detection, EdU is detected using a click reaction—a copper(I)-catalyzed reaction between an azide and an alkyne.|
Researchers no longer have to worry about cross-reactivity. The anti-BrdU antibody clone, MoBU-1, displays no cross-reactivity with EdU used in the Click-iT® EdU assay, greatly simplifying the workflow and results.
|Figure 5. Dual pulse labeling of cell proliferation with BrdU and Click-iT® EdU detected by fluorescence microscopy. U2OS cells were pulsed with 10 µM EdU for 60 min, washed, and then treated with chloroquine.|
|Figure 6. Dual pulse labeling of cell proliferation with BrdU and Click-iT® EdU detected by flow cytometry. TF-1 erythroblast cells were pulsed with 20 µM EdU for 1 hr followed by 10 µM BrdU for 1 hr. The cells were fixed in ethanol and an acid denaturation method was used before labeling with anti-BrdU (clone MoBU-1) Alexa Fluor® 488 conjugate and Click-iT® EdU Alexa Fluor® azide. Data were collected with a BD™ LSR II flow cytometer using 488 nm excitation with a 530/30 nm bandpass filter, and 633 nm excitation with a 660/20 nm bandpass filter. Cells colored blue are negative for both EdU and BrdU; cells colored dark green are positive for both EdU and BrdU; cells colored red are positive for EdU but negative for BrdU; cells colored light green are negative for EdU but positive for BrdU.|
Extracellular signals play a role in cell cycle regulation. TGF-β1 has been shown to be anti-mitogenic and capable of blocking cell cycle progression through the G1 phase of the cell cycle. The biological activity of recombinant human TGF-β1 has been confirmed by measuring the TGF-β1 dose dependent inhibition of proliferation of mouse HT-2 cells stimulated with mouse IL-4.
|Invitrogen offers recombinant human TGF-β1 as well as a number of other cytokines and growth factors for studying growth factor–induced signaling events. |
|Recombinant Human TGF-β1||100 µg||PHG9211|| |
|Recombinant Human TGF-β1||10 µg||PHG9214|
Two key classes of regulatory molecules, cyclins and cyclin-dependent kinases (CDKs), determine the progression of cells through the cell cycle. Cyclins form the regulatory subunits, and CDKs the catalytic subunits of the activated hetrodimer. When activated by cyclins, CDKs phosphorylate different targets so the cell can enter the next phase of the cell cycle. Different cyclin–CDK combinations determine downstream protein targets and thus different phases in the cell cycle.
Upon receiving the extracellular signal, G1 cyclin–CDK complexes become active and initiate a downstream cascade of events, preparing the cell to go to S phase and start DNA replication. Other cyclin–CDK complexes trigger the next stages of the cell cycle. Any abnormality in this activation can trigger the apoptotic pathway, which involves caspase activation and marks the cell for death. Recent studies have indicated that caspases such as caspase-3 not only function in apoptosis, but also regulate the cell cycle.
|We offer more than 400 antibodies for studying the cell cycle, apoptosis, and related pathways. These antibodies include phosphorylation site–specific antibodies, total protein detection antibodies, cleavage-specific antibodies, and new ABfinity ™ antibodies. ABfinity™ antibodies are rabbit recombinant antibodies produced by cloning the protein-specific genes and expressing them in mammalian cells. The result is a highly specific antibody with consistent lot-to-lot performance. An example is CASP3 [D175] ABfinity™ Recombinant Antibody (Cat. No. 700182), which detects only the cleaved, activated form of caspase-3 in applications such as western blot, immunocytochemistry, immunohistochemistry, and flow cytometry.|
|Figure 7. Immunocytochemistry of A549 cells labeled with rabbit anti-CASP3 [D175]. A549 cells were labeled with rabbit anti-CASP3 [D175] (5 µg/mL) treated without (left) or with (right) staurosporine. Alexa Fluor® 488 goat anti–rabbit IgG at 1:1,000 was used as the secondary antibody.|
|Figure 8. Western blot of Jurkat lysates labeled with rabbit anti-CASP3 [D175]. Rabbit anti-CASP3 [D175] (0.1 µg/mL) was used to label cleaved caspase-3 in untreated Jurkat lysates (lane 1) or staurosporin-treated Jurkat lysates (lane 2).|
|Target Protein||Reactive Species: Tested (Expected)||Applications||Quantity||Cat. No.|
|CASP3 [D175]||Hu (B, Cn, Cp, Eq, Fe, Ha, Ms, P, R, Rt, Sh, Sw, X)||E, F, IF/ICC, IHC,WB||100 µg||700182|
|Cyclin D1||Hu, Ms, Rt||IHC, IF, IP (co), KA, WB||500 µg||AHF0102|
|Cdk1 [pT14/pY15]||Hu, Ms, Rt||IP, WB||10 blot||44686G|
|Cyclin E||Hu||FC, IHC, IP, WB||200 µg||321600|
|Cyclin B1||Ha, Hu, Ms, Rt||FC, IHC, IP (co), WB||100 µg||AHF0052|
|Reactivity: B = bovine; Cn = canine; Cp = chimpanzee; Eq = equine; Fe = feline; Ha = hamster; Hu = human; Ms = mouse; P = porcine; R = rabbit; Rt = rat; Sh = sheep; Sw = swine; X = Xenopus.|
Applications: E = ELISA; F = fluorescence; FC = flow cytometry; ICC = immunocytochemistry; IF = immunofluorescence; IHC = immunohistochemistry; IP = immunoprecipitation; WB = western blotting.