Description
PBK/TOPK (Phospho-Thr9)Colorimetric Cell-Based ELISA Kit
The PBK (P21-Activated Kinase) Colorimetric Cell-Based ELISA Kit is specifically designed for the detection of phosphorylated Thr9 on the PBK/Topk protein in cell lysates. This kit offers high sensitivity and accuracy, providing researchers with reliable and reproducible results for studying the activation state of PBK/Topk in various cell types.PBK/Topk is a serine/threonine kinase known to play a role in cell proliferation, migration, and survival. Phosphorylation of Thr9 on PBK/Topk has been linked to various cellular processes and diseases, including cancer and neurodegenerative disorders.
By accurately measuring phosphorylated Thr9 levels, researchers can gain valuable insights into the role of PBK/Topk in these conditions and potentially uncover new therapeutic targets.Overall, the PBK/Topk Phospho-Thr9 Colorimetric Cell-Based ELISA Kit is a valuable tool for studying the signaling pathways involving PBK/Topk and understanding its implications in disease development and progression. Its ease of use and reliable performance make it suitable for a wide range of research applications in the fields of cancer biology, neurobiology, and drug discovery.
| Product Name: | PBK/TOPK (Phospho-Thr9)Colorimetric Cell-Based ELISA Kit |
| Product Code: | CBCAB01353 |
| ELISA Type: | Cell-Based |
| Target: | PBK/TOPK (Phospho-Thr9) |
| Reactivity: | Human |
| Dynamic Range: | > 5000 Cells |
| Detection Method: | Colorimetric 450 nm |
| Format: | 2 x 96-Well Microplates |
The PBK/TOPK (Phospho-Thr9) Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect PBK/TOP protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated PBK/TOP in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on PBK/TOP phosphorylation.
Qualitative determination of PBK/TOPK (Phospho-Thr9) concentration is achieved by an indirect ELISA format. In essence, PBK/TOPK (Phospho-Thr9) is captured by PBK/TOPK (Phospho-Thr9)-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: 55872, UniProt ID: Q96KB5, OMIM: 611210, Unigene: Hs.104741 |
| Gene Symbol: | PBK |
| Sub Type: | Phospho |
| UniProt Protein Function: | PBK: Phosphorylates MAP kinase p38. Seems to be active only in mitosis. May also play a role in the activation of lymphoid cells. When phosphorylated, forms a complex with TP53, leading to TP53 destabilization and attenuation of G2/M checkpoint during doxorubicin-induced DNA damage. Interacts with DLG1 and TP53. Expressed in the testis and placenta. In the testis, restrictedly expressed in outer cell layer of seminiferous tubules. Activated by phosphorylation. Belongs to the protein kinase superfamily. Ser/Thr protein kinase family. MAP kinase kinase subfamily. |
| UniProt Protein Details: | Protein type:EC 2.7.12.2; Cancer Testis Antigen (CTA); Protein kinase, Ser/Thr (non-receptor); Kinase, protein; Protein kinase, Other; Other group; TOPK family Chromosomal Location of Human Ortholog: 8p21.2 Cellular Component: nucleus Molecular Function:ATP binding; protein binding; protein serine/threonine kinase activity Biological Process: mitosis; negative regulation of inflammatory response; negative regulation of proteasomal ubiquitin-dependent protein catabolic process; negative regulation of stress-activated MAPK cascade; protein amino acid phosphorylation |
| NCBI Summary: | This gene encodes a serine/threonine protein kinase related to the dual specific mitogen-activated protein kinase kinase (MAPKK) family. Evidence suggests that mitotic phosphorylation is required for its catalytic activity. The encoded protein may be involved in the activation of lymphoid cells and support testicular functions, with a suggested role in the process of spermatogenesis. Overexpression of this gene has been implicated in tumorigenesis. Alternative splicing results in multiple transcript variants. [provided by RefSeq, Jul 2013] |
| UniProt Code: | Q96KB5 |
| NCBI GenInfo Identifier: | 83305809 |
| NCBI Gene ID: | 55872 |
| NCBI Accession: | Q96KB5.3 |
| UniProt Secondary Accession: | Q96KB5,Q9NPD9, Q9NYL7, Q9NZK6, B4DX68, D3DST2, |
| UniProt Related Accession: | Q96KB5 |
| Molecular Weight: | 37,211 Da |
| NCBI Full Name: | Lymphokine-activated killer T-cell-originated protein kinase |
| NCBI Synonym Full Names: | PDZ binding kinase |
| NCBI Official Symbol: | PBK |
| NCBI Official Synonym Symbols: | SPK; CT84; TOPK; HEL164; Nori-3 |
| NCBI Protein Information: | lymphokine-activated killer T-cell-originated protein kinase |
| UniProt Protein Name: | Lymphokine-activated killer T-cell-originated protein kinase |
| UniProt Synonym Protein Names: | Cancer/testis antigen 84; CT84; MAPKK-like protein kinase; Nori-3; PDZ-binding kinase; Spermatogenesis-related protein kinase; SPK; T-LAK cell-originated protein kinase |
| Protein Family: | Lymphokine-activated killer T-cell-originated protein kinase |
| UniProt Gene Name: | PBK |
| UniProt Entry Name: | TOPK_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-PBK/TOPK (Phospho-Thr9) Antibody, Anti-PBK/TOPK 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.)
