Description
DAPK3 (Phospho-Thr265)Colorimetric Cell-Based ELISA Kit
The DAPK3 (Death-Associated Protein Kinase 3) Phospho-Thr265 Colorimetric Cell-Based ELISA Kit is specifically designed for the quantitative detection of DAPK3 phosphorylated at threonine 265 in cell lysates. This kit offers high sensitivity and specificity, allowing for accurate and reliable measurement of DAPK3 activity in various cell types.DAPK3 is a kinase that plays a key role in cell death and survival processes, making it a crucial target for studying cellular signaling pathways and potential therapeutic interventions.
Phosphorylation of DAPK3 at threonine 265 has been implicated in regulating its enzymatic activity and cellular functions.By using the DAPK3 Phospho-Thr265 Colorimetric Cell-Based ELISA Kit, researchers can gain valuable insights into the role of DAPK3 in various biological processes and diseases, such as cancer, neurodegeneration, and inflammation. This kit provides a powerful tool for studying DAPK3 signaling pathways and developing novel therapies targeting this important kinase.
| Product Name: | DAPK3 (Phospho-Thr265)Colorimetric Cell-Based ELISA Kit |
| Product Code: | CBCAB01414 |
| ELISA Type: | Cell-Based |
| Target: | DAPK3 (Phospho-Thr265) |
| Reactivity: | Human, Mouse, Rat |
| Dynamic Range: | > 5000 Cells |
| Detection Method: | Colorimetric 450 nm |
| Format: | 2 x 96-Well Microplates |
The DAPK3 (Phospho-Thr265) Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect DAPK3 protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated DAPK3 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on DAPK3 phosphorylation.
Qualitative determination of DAPK3 (Phospho-Thr265) concentration is achieved by an indirect ELISA format. In essence, DAPK3 (Phospho-Thr265) is captured by DAPK3 (Phospho-Thr265)-specific primary (1ø) antibodies while the HRP-conjugated secondary (2ø) antibodies bind the Fc region of the 1ø antibody. Through this binding, the HRP enzyme conjugated to the 2ø antibody can catalyze a colorimetric reaction upon substrate addition. Due to the qualitative nature of the Cell-Based ELISA, multiple normalization methods are needed:
| 1. | A monoclonal antibody specific for human GAPDH is included to serve as an internal positive control in normalizing the target absorbance values. |
| 2. | Following the colorimetric measurement of HRP activity via substrate addition, the Crystal Violet whole-cell staining method may be used to determine cell density. After staining, the results can be analysed by normalizing the absorbance values to cell amounts, by which the plating difference can be adjusted. |
| Database Information: | Gene ID: 1613, UniProt ID: O43293, OMIM: 603289, Unigene: Hs.631844 |
| Gene Symbol: | DAPK3 |
| Sub Type: | Phospho |
| UniProt Protein Function: | DAPK3: Serine/threonine kinase which is involved in the regulation of apoptosis, autophagy, transcription, actin cytoskeleton reorganization, cell motility, smooth muscle contraction, and mitosis, particularly cytokinesis. Regulates both type I apoptotic and type II autophagic cell deaths signal, depending on the cellular setting. The former is caspase- dependent, while the latter is caspase-independent and is characterized by the accumulation of autophagic vesicles. Regulates myosin phosphorylation in both smooth muscle and non- muscle cells. In smooth muscle, regulates myosin either directly by phosphorylating MYL12B and MYL9 or through inhibition of smooth muscle myosin phosphatase (SMPP1M) via phosphorylation of PPP1R12A, and the inhibition of SMPP1M functions to enhance muscle responsiveness to Ca(2+) and promote a contractile state. Enhances transcription from AR-responsive promoters in a hormone- and kinase-dependent manner. Phosphorylates STAT3 and enhances its transcriptional activity. Positively regulates the canonical Wnt/beta-catenin signaling through interaction with NLK and TCF7L2. Can disrupt the NLK-TCF7L2 complex thereby influencing the phosphorylation of TCF7L2 by NLK. Phosphorylates histone H3 on 'Thr-11' at centromeres during mitosis. Involved in the formation of promyelocytic leukemia protein nuclear body (PML-NB), one of many subnuclear domains in the eukaryotic cell nucleus, and which is involved in oncogenesis and viral infection. Monomer and homotrimer. Can also exist as homodimer or form heterodimers with ATF4. Homodimerization is required for activation segment autophosphorylation Both interactions require an intact leucine zipper domain and oligomerization is required for full enzymatic activity. Also binds to DAXX and PAWR, possibly in a ternary complex which plays a role in caspase activation. According to PubMed:17953487, does not interact with PARW. Interacts with AATF, CDC5L, UBE2D1, UBE2D2 AND UBE2D3. Interacts with AR and this interaction is enhanced by AATF. Interacts (via leucine zipper) with TCP10L (via leucine zipper). Interacts (via kinase domain) with DAPK1 (via kinase domain).Interacts with STAT3, NLK and TCF7L2. Isoform 1 and isoform 2 can interact with myosin and PPP1R12A. Isoform 2 is expressed in the bladder smooth muscle. Inhibited by pyridone 6 (K00225), a potent, ATP-competitive inhibitor. Phosphorylation at Thr-180, Thr-225 and Thr-265 is essential for activity. Oligomerization is required for full enzymatic activity. Belongs to the protein kinase superfamily. CAMK Ser/Thr protein kinase family. DAP kinase subfamily. 2 isoforms of the human protein are produced by alternative splicing. |
| UniProt Protein Details: | Protein type:Kinase, protein; Protein kinase, Ser/Thr (non-receptor); Tumor suppressor; Protein kinase, CAMK; EC 2.7.11.1; CAMK group; DAPK family Chromosomal Location of Human Ortholog: 19p13.3 Cellular Component: PML body; cytoplasm; nucleus; lipid raft Molecular Function:identical protein binding; protein serine/threonine kinase activity; leucine zipper domain binding; protein binding; protein homodimerization activity; Rho GTPase binding; calmodulin-dependent protein kinase activity; cAMP response element binding protein binding; ATP binding Biological Process: regulation of smooth muscle contraction; transcription, DNA-dependent; positive regulation of apoptosis; apoptosis; protein amino acid autophosphorylation; regulation of myosin II filament assembly or disassembly; regulation of mitotic cell cycle; cytokinesis; chromatin modification; protein amino acid phosphorylation; regulation of apoptosis; neuron differentiation; regulation of cell shape; regulation of transcription, DNA-dependent; negative regulation of translation; regulation of focal adhesion formation; regulation of mitosis; regulation of autophagy; positive regulation of cell migration |
| NCBI Summary: | Death-associated protein kinase 3 (DAPK3) induces morphological changes in apoptosis when overexpressed in mammalian cells. These results suggest that DAPK3 may play a role in the induction of apoptosis. [provided by RefSeq, Jul 2008] |
| UniProt Code: | O43293 |
| NCBI GenInfo Identifier: | 4557511 |
| NCBI Gene ID: | 1613 |
| NCBI Accession: | NP_001339.1 |
| UniProt Related Accession: | O43293 |
| Molecular Weight: | ~ 52kDa |
| NCBI Full Name: | death-associated protein kinase 3 |
| NCBI Synonym Full Names: | death associated protein kinase 3 |
| NCBI Official Symbol: | DAPK3 |
| NCBI Official Synonym Symbols: | DLK; ZIP; ZIPK |
| NCBI Protein Information: | death-associated protein kinase 3 |
| UniProt Protein Name: | Death-associated protein kinase 3 |
| UniProt Synonym Protein Names: | DAP-like kinase; Dlk; MYPT1 kinase; Zipper-interacting protein kinase |
| Protein Family: | Myosin |
| UniProt Gene Name: | DAPK3 |
| UniProt Entry Name: | DAPK3_HUMAN |
| Component | Quantity |
| 96-Well Cell Culture Clear-Bottom Microplate | 2 plates |
| 10X TBS | 24 mL |
| Quenching Buffer | 24 mL |
| Blocking Buffer | 50 mL |
| 15X Wash Buffer | 50 mL |
| Primary Antibody Diluent | 12 mL |
| 100x Anti-Phospho Target Antibody | 60 µL |
| 100x Anti-Target Antibody | 60 µL |
| Anti-GAPDH Antibody | 60 µL |
| HRP-Conjugated Anti-Rabbit IgG Antibody | 12 mL |
| HRP-Conjugated Anti-Mouse IgG Antibody | 12 mL |
| SDS Solution | 12 mL |
| Stop Solution | 24 mL |
| Ready-to-Use Substrate | 12 mL |
| Crystal Violet Solution | 12 mL |
| Adhesive Plate Seals | 2 seals |
The following materials and/or equipment are NOT provided in this kit but are necessary to successfully conduct the experiment:
- Microplate reader able to measure absorbance at 450 nm and/or 595 nm for Crystal Violet Cell Staining (Optional)
- Micropipettes with capability of measuring volumes ranging from 1 µL to 1 ml
- 37% formaldehyde (Sigma Cat# F-8775) or formaldehyde from other sources
- Squirt bottle, manifold dispenser, multichannel pipette reservoir or automated microplate washer
- Graph paper or computer software capable of generating or displaying logarithmic functions
- Absorbent papers or vacuum aspirator
- Test tubes or microfuge tubes capable of storing ≥1 ml
- Poly-L-Lysine (Sigma Cat# P4832 for suspension cells)
- Orbital shaker (optional)
- Deionized or sterile water
*Note: Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.
| Step | Procedure |
| 1. | Seed 200 µL of 20,000 adherent cells in culture medium in each well of a 96-well plate. The plates included in the kit are sterile and treated for cell culture. For suspension cells and loosely attached cells, coat the plates with 100 µL of 10 µg/ml Poly-L-Lysine (not included) to each well of a 96-well plate for 30 minutes at 37 °C prior to adding cells. |
| 2. | Incubate the cells for overnight at 37 °C, 5% CO2. |
| 3. | Treat the cells as desired. |
| 4. | Remove the cell culture medium and rinse with 200 µL of 1x TBS, twice. |
| 5. | Fix the cells by incubating with 100 µL of Fixing Solution for 20 minutes at room temperature. The 4% formaldehyde is used for adherent cells and 8% formaldehyde is used for suspension cells and loosely attached cells. |
| 6. | Remove the Fixing Solution and wash the plate 3 times with 200 µL 1x Wash Buffer for five minutes each time with gentle shaking on the orbital shaker. The plate can be stored at 4 °C for a week. |
| 7. | Add 100 µL of Quenching Buffer and incubate for 20 minutes at room temperature. |
| 8. | Wash the plate 3 times with 1x Wash Buffer for 5 minutes each time. |
| 9. | Add 200 µL of Blocking Buffer and incubate for 1 hour at room temperature. |
| 10. | Wash 3 times with 200 µL of 1x Wash Buffer for 5 minutes each time. |
| 11. | Add 50 µL of 1x primary antibodies Anti-DAPK3 (Phospho-Thr265) Antibody, Anti-DAPK3 Antibody and/or Anti-GAPDH Antibody) to the corresponding wells, cover with Parafilm and incubate for 16 hours (overnight) at 4 °C. If the target expression is known to be high, incubate for 2 hours at room temperature. |
| 12. | Wash 3 times with 200 µL of 1x Wash Buffer for 5 minutes each time. |
| 13. | Add 50 µL of 1x secondary antibodies (HRP-Conjugated AntiRabbit IgG Antibody or HRP-Conjugated Anti-Mouse IgG Antibody) to corresponding wells and incubate for 1.5 hours at room temperature. |
| 14. | Wash 3 times with 200 µL of 1x Wash Buffer for 5 minutes each time. |
| 15. | Add 50 µL of Ready-to-Use Substrate to each well and incubate for 30 minutes at room temperature in the dark. |
| 16. | Add 50 µL of Stop Solution to each well and read OD at 450 nm immediately using the microplate reader. |
(Additional Crystal Violet staining may be performed if desired – details of this may be found in the kit technical manual.)
