Cell Cycle and Apoptosis WB Cocktail (pCdk/pHH3/Actin/PARP) (ab139417) is part of the reagents, controls & accessories range. Abcam offers high-quality biological reagents and tools including antibodies, proteins, assays, cell lines and lysates.
Application | Reactivity | Dilution info | Notes |
---|---|---|---|
Application WB | Reactivity Reacts | Dilution info - | Notes 1/250 dilution for primary antibody cocktail. 1/2500 dilution for secondary antibody HRP cocktail. |
Cell Cycle and Apoptosis WB Cocktail (pCdk/pHH3/Actin/PARP) (ab139417) is part of the reagents, controls & accessories range. Abcam offers high-quality biological reagents and tools including antibodies, proteins, assays, cell lines and lysates.
Preservative: 0.01% Sodium azide
Constituents: 50% Glycerol (glycerin, glycerine), 0.87% Sodium chloride, 0.79% Tris HCl, 0.05% BSA
The Cell Cycle & apoptosis western blot cocktail (ab139417) is designed to provide a rapid assessment of cell cycle distribution and apoptosis induction based on molecular markers for the G1/S and M phases of the cell cycle and the presence of cleaved PARP. This is a mixture of four highly specific monoclonal primary antibodies targeting Cdk2 pTyr15, Histone H3 pSer10 , cleaved-PARP and Actin. Cyclin-dependent kinase 2 (Cdk2) is maintained in an inactive state in G1/S by inhibitory phosphorylation on Tyr15. Histone H3 is phosphorylated at Ser10 when chromosomes condense during mitosis. Hence, elevated Cdk2 pTyr15 indicates G1/S arrested cells and elevated Histone H3 pSer10 indicates M-phase arrested cells when compared to asynchronous cycling control cells. Poly [ADP-ribose] polymerase 1 (PARP) is a DNA repair enzyme that is cleaved by activated caspases, hence cleaved-PARP indicates induction of apoptosis. An anti- Actin antibody is included as a loading control. These four readouts are easily resolved by western blot given their different molecular weights. Because this western blot cocktail is a mix of rabbit and mouse monoclonal primary antibodies, a secondary antibody cocktail of HRP conjugated antibodies (anti-mouse and anti-rabbit) is provided.
The biological target 'Muscle Actin+Histone H3+Cdk2+Cleaved PARP' comprises various proteins with distinct functions. Muscle actin a part of the actin family plays an important role in muscle contraction and is expressed in muscle cells with a molecular weight of about 42 kDa. Histone H3 a core component of chromatin in the nuclei of eukaryotic cells has a mass of around 15 kDa and is important in the compact structure of chromosomal DNA. Cyclin-dependent kinase 2 (Cdk2) is essential for cell cycle regulation and weighs approximately 34 kDa found in the nucleus of proliferating cells. Cleaved PARP (Poly ADP-ribose polymerase) is a 116 kDa protein involved in DNA repair and is expressed in various tissues particularly during apoptosis where it gets cleaved into 89 kDa and 24 kDa fragments.
These proteins have diverse roles within cellular processes. Muscle actin contributes to cytoskeletal integrity and cell motility. Histone H3 is involved in the regulation of gene expression through chromatin remodeling. Cdk2 partners with cyclins to progress the cell cycle especially during the S and G2 phases. Cleaved PARP is important in apoptosis indicating cells undergoing programmed cell death. These proteins may participate in complexes such as the nucleosome for Histone H3 or with cyclins in cell cycle regulation for Cdk2.
These proteins play significant parts in different biological pathways. Cdk2 is an important player in the cell cycle pathway working with cyclins like Cyclin E and Cyclin A to control transitions between cell cycle phases. Cleaved PARP participates in the apoptosis pathway interacting with caspases that execute cell demise. These pathways illustrate the intricate network of interactions with proteins like retinoblastoma protein (Rb) associating with Cdk2 for cell cycle regulation.
These proteins are linked to various medical conditions. Aberrations in Cdk2 activity often relate to cancer as uncontrolled cell proliferation occurs. In neurodegenerative diseases like Alzheimer's the regulation of apoptosis through cleaved PARP points to its role in neuronal cell death. In cancer PARP is often associated with BRCA1 and BRCA2 genes connected to breast and ovarian cancers highlighting the importance of DNA repair in oncogenesis.
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Terms & Conditions.
Paclitaxel induces dose-dependent G2/M-phase arrest and induces apoptosis (decreased Cdk2 pTyr15, increased Histone H3 pSer10, cleaved PARP present).
Primary antibody: All lanes Cell Cycle WB cocktail (MS1123) diluted to 1X in 4% milk/PBS.
Lane 1: HeLa lysate; Untreated, asynchronous cells
Lane 2: HeLa lysate; 2000 nM Paclitaxel, 24h
Lane 3: HeLa lysate; 100 nM Paclitaxel, 24h
Lane 4: HeLa lysate; 5 nM Paclitaxel, 24h
Lane 5: HeLa lysate; 0.25 nM Paclitaxel, 24h
All lysates at 15 µg per lane. Lysates are HeLa cells lysed in SDS lysis buffer.
Secondary antibody: All lanes are the WB secondary antibody HRP cocktail diluted to 1X in 4% milk/PBS.
Hydroxyurea induces a G1/S-phase cell cycle arrest and induces apoptosis (increased Cdk2 pTyr15, decreased Histone H3 pSer10, cleaved PARP present). Paclitaxel induces dose-dependent G2/M-phase arrest and induces apoptosis (decreased Cdk2 pTyr15, increased Histone H3 pSer10, cleaved PARP present).
Primary antibody: All lanes Cell Cycle WB cocktail (ab139417) diluted to 1X in 4% milk/PBS.
Lane 1: Jurkat lysate; Untreated, asynchronous cells
Lane 2: Jurkat lysate; 5 mM Hydroxyurea, 24h
Lane 3: Jurkat lysate; 1000 nM Paclitaxel, 24h
Lane 4: Jurkat lysate; 50 nM Paclitaxel, 24h
Lane 5: Jurkat lysate; 2.5 nM Paclitaxel, 24h
All lysates at 12 µg per lane. Lysates are Jurkat cells lysed in SDS lysis buffer.
Secondary antibody: All lanes are the WB secondary antibody HRP cocktail diluted to 1X in 4% milk/PBS.
Predicted & observed band size: 89, 42, 33, 17 kDa.
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