Human Cyclin-dependent kinase 1 (CDK1) ELISA Kit
- SKU:
- HUEB2014
- Product Type:
- ELISA Kit
- Size:
- 96 Assays
- Uniprot:
- P06493
- ELISA Type:
- Sandwich
- Synonyms:
- CDK1, CDC28A, cell cycle controller CDC2, Cell division control protein 2 homolog, Cell division protein kinase 1, cyclin-dependent kinase 1, G1 to S and G2 to M, p34 protein kinase, P34CDC2
- Reactivity:
- Human
Description
| Product Name: | Human Cyclin-dependent kinase 1 (CDK1) ELISA Kit |
| Product Code: | HUEB2014 |
| Alias: | Cyclin-dependent kinase 1, CDK1, Cell division control protein 2 homolog, Cell division protein kinase 1, p34 protein kinase, CDK1, CDC2, CDC28A, CDKN1, P34CDC2, 2.7.11.22 |
| Uniprot: | P06493 |
| Reactivity: | Human |
| Range: | Please contact us for more information |
| Detection Method: | Sandwich |
| Size: | 96 Assay |
| Storage: | Please see kit components below for exact storage details |
| Note: | For research use only |
| UniProt Protein Function: | CDK1: a protein kinase of the CDK family. Catalytic subunit of the conserved protein complex known as M-phase promoting factor (MPF), which is essential for G1/S and G2/M phase transitions. Mitotic cyclins stably associate with this protein and function as regulatory subunits. Its activity is controlled by cyclin availability and phosphorylation through the cell cycle. Activated in many cancers including colon, liver and breast. The T isoform, which lacks a regulatory region, is expressed in breast cancer. Inhibition in cancer cells may drive cells into apoptosis. May also drive cell migration. Inhibitors: BMS-265246, BMS-265246-01. 2 isoforms of the human protein are produced by alternative splicing. |
| UniProt Protein Details: | Protein type:Cell cycle regulation; EC 2.7.11.23; EC 2.7.11.22; Motility/polarity/chemotaxis; Kinase, protein; Protein kinase, CMGC; Protein kinase, Ser/Thr (non-receptor); CMGC group; CDK family; CDK/CDK1 subfamily; CDK1 subfamily Chromosomal Location of Human Ortholog: 10q21.1 Cellular Component: nucleoplasm; centrosome; membrane; mitochondrion; spindle microtubule; cytoplasm; midbody; nucleus; cytosol Molecular Function:RNA polymerase subunit kinase activity; protein serine/threonine kinase activity; protein binding; cyclin binding; cyclin-dependent protein kinase activity; histone kinase activity; Hsp70 protein binding; ATP binding; protein kinase activity Biological Process: regulation of Schwann cell differentiation; activation of MAPKK activity; nerve growth factor receptor signaling pathway; activation of MAPK activity; histone phosphorylation; response to toxin; ventricular cardiac muscle cell development; regulation of embryonic development; centrosome cycle; stress-activated MAPK cascade; toll-like receptor 3 signaling pathway; response to organic cyclic substance; toll-like receptor 5 signaling pathway; small GTPase mediated signal transduction; protein complex assembly; G2/M transition of mitotic cell cycle; toll-like receptor 4 signaling pathway; positive regulation of cardiac muscle cell proliferation; response to drug; mitosis; organ regeneration; fibroblast growth factor receptor signaling pathway; positive regulation of protein import into nucleus, translocation; cell aging; pronuclear fusion; toll-like receptor 2 signaling pathway; chromosome condensation; response to ethanol; cell division; response to activity; toll-like receptor 9 signaling pathway; response to amine stimulus; G1/S transition of mitotic cell cycle; negative regulation of apoptosis; positive regulation of ubiquitin-protein ligase activity during mitotic cell cycle; axon guidance; apoptosis; mitotic nuclear envelope disassembly; positive regulation of mitotic cell cycle; toll-like receptor 10 signaling pathway; epithelial cell differentiation; anaphase-promoting complex-dependent proteasomal ubiquitin-dependent protein catabolic process; response to axon injury; DNA replication; epidermal growth factor receptor signaling pathway; cell migration; MyD88-independent toll-like receptor signaling pathway; organelle organization and biogenesis; MAPKKK cascade; peptidyl-threonine phosphorylation; microtubule cytoskeleton organization and biogenesis; DNA repair; MyD88-dependent toll-like receptor signaling pathway; G1/S-specific transcription in mitotic cell cycle; peptidyl-serine phosphorylation; response to cadmium ion; regulation of ubiquitin-protein ligase activity during mitotic cell cycle; response to copper ion; mitotic cell cycle G2/M transition DNA damage checkpoint; Ras protein signal transduction; insulin receptor signaling pathway; toll-like receptor signaling pathway; innate immune response; mitotic cell cycle; vascular endothelial growth factor receptor signaling pathway; positive regulation of DNA replication |
| NCBI Summary: | The protein encoded by this gene is a member of the Ser/Thr protein kinase family. This protein is a catalytic subunit of the highly conserved protein kinase complex known as M-phase promoting factor (MPF), which is essential for G1/S and G2/M phase transitions of eukaryotic cell cycle. Mitotic cyclins stably associate with this protein and function as regulatory subunits. The kinase activity of this protein is controlled by cyclin accumulation and destruction through the cell cycle. The phosphorylation and dephosphorylation of this protein also play important regulatory roles in cell cycle control. Alternatively spliced transcript variants encoding different isoforms have been found for this gene. [provided by RefSeq, Mar 2009] |
| UniProt Code: | P06493 |
| NCBI GenInfo Identifier: | 334302921 |
| NCBI Gene ID: | 983 |
| NCBI Accession: | P06493.3 |
| UniProt Secondary Accession: | P06493,O60764, A8K7C4, C9J497, |
| UniProt Related Accession: | P06493 |
| Molecular Weight: | 297 |
| NCBI Full Name: | Cyclin-dependent kinase 1 |
| NCBI Synonym Full Names: | cyclin-dependent kinase 1 |
| NCBI Official Symbol: | CDK1Â Â |
| NCBI Official Synonym Symbols: | CDC2; CDC28A; P34CDC2Â Â |
| NCBI Protein Information: | cyclin-dependent kinase 1; p34 protein kinase; cell cycle controller CDC2; cell division protein kinase 1; cell division control protein 2 homolog; cell division cycle 2, G1 to S and G2 to M |
| UniProt Protein Name: | Cyclin-dependent kinase 1 |
| UniProt Synonym Protein Names: | Cell division control protein 2 homolog; Cell division protein kinase 1; p34 protein kinase |
| UniProt Gene Name: | CDK1Â Â |
| UniProt Entry Name: | CDK1_HUMAN |
| Component | Quantity (96 Assays) | Storage |
| ELISA Microplate (Dismountable) | 8×12 strips | -20°C |
| Lyophilized Standard | 2 | -20°C |
| Sample Diluent | 20ml | -20°C |
| Assay Diluent A | 10mL | -20°C |
| Assay Diluent B | 10mL | -20°C |
| Detection Reagent A | 120µL | -20°C |
| Detection Reagent B | 120µL | -20°C |
| Wash Buffer | 30mL | 4°C |
| Substrate | 10mL | 4°C |
| Stop Solution | 10mL | 4°C |
| Plate Sealer | 5 | - |
Other materials and equipment required:
- Microplate reader with 450 nm wavelength filter
- Multichannel Pipette, Pipette, microcentrifuge tubes and disposable pipette tips
- Incubator
- Deionized or distilled water
- Absorbent paper
- Buffer resevoir
*Note: The below protocol is a sample protocol. Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.
Allow all reagents to reach room temperature (Please do not dissolve the reagents at 37°C directly). All the reagents should be mixed thoroughly by gently swirling before pipetting. Avoid foaming. Keep appropriate numbers of strips for 1 experiment and remove extra strips from microtiter plate. Removed strips should be resealed and stored at -20°C until the kits expiry date. Prepare all reagents, working standards and samples as directed in the previous sections. Please predict the concentration before assaying. If values for these are not within the range of the standard curve, users must determine the optimal sample dilutions for their experiments. We recommend running all samples in duplicate.
| Step | |
| 1. | Add Sample: Add 100µL of Standard, Blank, or Sample per well. The blank well is added with Sample diluent. Solutions are added to the bottom of micro ELISA plate well, avoid inside wall touching and foaming as possible. Mix it gently. Cover the plate with sealer we provided. Incubate for 120 minutes at 37°C. |
| 2. | Remove the liquid from each well, don't wash. Add 100µL of Detection Reagent A working solution to each well. Cover with the Plate sealer. Gently tap the plate to ensure thorough mixing. Incubate for 1 hour at 37°C. Note: if Detection Reagent A appears cloudy warm to room temperature until solution is uniform. |
| 3. | Aspirate each well and wash, repeating the process three times. Wash by filling each well with Wash Buffer (approximately 400µL) (a squirt bottle, multi-channel pipette,manifold dispenser or automated washer are needed). Complete removal of liquid at each step is essential. After the last wash, completely remove remaining Wash Buffer by aspirating or decanting. Invert the plate and pat it against thick clean absorbent paper. |
| 4. | Add 100µL of Detection Reagent B working solution to each well. Cover with the Plate sealer. Incubate for 60 minutes at 37°C. |
| 5. | Repeat the wash process for five times as conducted in step 3. |
| 6. | Add 90µL of Substrate Solution to each well. Cover with a new Plate sealer and incubate for 10-20 minutes at 37°C. Protect the plate from light. The reaction time can be shortened or extended according to the actual color change, but this should not exceed more than 30 minutes. When apparent gradient appears in standard wells, user should terminatethe reaction. |
| 7. | Add 50µL of Stop Solution to each well. If color change does not appear uniform, gently tap the plate to ensure thorough mixing. |
| 8. | Determine the optical density (OD value) of each well at once, using a micro-plate reader set to 450 nm. User should open the micro-plate reader in advance, preheat the instrument, and set the testing parameters. |
| 9. | After experiment, store all reagents according to the specified storage temperature respectively until their expiry. |
When carrying out an ELISA assay it is important to prepare your samples in order to achieve the best possible results. Below we have a list of procedures for the preparation of samples for different sample types.
| Sample Type | Protocol |
| Serum | If using serum separator tubes, allow samples to clot for 30 minutes at room temperature. Centrifuge for 10 minutes at 1,000x g. Collect the serum fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. If serum separator tubes are not being used, allow samples to clot overnight at 2-8°C. Centrifuge for 10 minutes at 1,000x g. Remove serum and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. |
| Plasma | Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples at 4°C for 15 mins at 1000 × g within 30 mins of collection. Collect the plasma fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. Note: Over haemolysed samples are not suitable for use with this kit. |
| Urine & Cerebrospinal Fluid | Collect the urine (mid-stream) in a sterile container, centrifuge for 20 mins at 2000-3000 rpm. Remove supernatant and assay immediately. If any precipitation is detected, repeat the centrifugation step. A similar protocol can be used for cerebrospinal fluid. |
| Cell culture supernatant | Collect the cell culture media by pipette, followed by centrifugation at 4°C for 20 mins at 1500 rpm. Collect the clear supernatant and assay immediately. |
| Cell lysates | Solubilize cells in lysis buffer and allow to sit on ice for 30 minutes. Centrifuge tubes at 14,000 x g for 5 minutes to remove insoluble material. Aliquot the supernatant into a new tube and discard the remaining whole cell extract. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C. |
| Tissue homogenates | The preparation of tissue homogenates will vary depending upon tissue type. Rinse tissue with 1X PBS to remove excess blood & homogenize in 20ml of 1X PBS (including protease inhibitors) and store overnight at ≤ -20°C. Two freeze-thaw cycles are required to break the cell membranes. To further disrupt the cell membranes you can sonicate the samples. Centrifuge homogenates for 5 mins at 5000xg. Remove the supernatant and assay immediately or aliquot and store at -20°C or -80°C. |
| Tissue lysates | Rinse tissue with PBS, cut into 1-2 mm pieces, and homogenize with a tissue homogenizer in PBS. Add an equal volume of RIPA buffer containing protease inhibitors and lyse tissues at room temperature for 30 minutes with gentle agitation. Centrifuge to remove debris. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C. |
| Breast Milk | Collect milk samples and centrifuge at 10,000 x g for 60 min at 4°C. Aliquot the supernatant and assay. For long term use, store samples at -80°C. Minimize freeze/thaw cycles. |
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