Description
Human Dynamin-2 (DNM2) ELISA Kit
The Human Dynamin 2 (DNM2) ELISA Kit is a powerful tool for measuring levels of dynamin 2 in human samples such as serum, plasma, and cell culture supernatants. With its high sensitivity and specificity, this kit provides accurate and reliable results for a variety of research applications.Dynamin 2 is a key protein involved in cellular processes such as endocytosis and membrane trafficking. Dysregulation of dynamin 2 has been linked to various diseases including Charcot-Marie-Tooth disease and centronuclear myopathy.
Therefore, measuring dynamin 2 levels can help researchers better understand the underlying mechanisms of these disorders and aid in the development of potential therapeutic interventions.Overall, the Human Dynamin 2 (DNM2) ELISA Kit is a valuable tool for researchers studying dynamin 2 biology and its implications in human health and disease.
| Product Name: | Human Dynamin-2 (DNM2) ELISA Kit |
| SKU: | HUEB1196 |
| Size: | 96T |
| Target: | Human Dynamin-2 (DNM2) |
| Synonyms: | DYN2 |
| Assay Type: | Sandwich |
| Detection Method: | ELISA |
| Reactivity: | Human |
| Detection Range: | 15.6-1000pg/mL |
| Sensitivity: | 10pg/mL |
| Intra CV: | 5.2% | ||||||||||||||||||||
| Inter CV: | 9.4% | ||||||||||||||||||||
| Linearity: |
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| Recovery: |
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| Function: | Microtubule-associated force-producing protein involved in producing microtubule bundles and able to bind and hydrolyze GTP. Plays a role in the regulation of neuron morphology, axon growth and formation of neuronal growth cones (By similarity). Plays an important role in vesicular trafficking processes, in particular endocytosis. Involved in cytokinesis (PubMed:12498685). Regulates maturation of apoptotic cell corpse-containing phagosomes by recruiting PIK3C3 to the phagosome membrane. |
| Uniprot: | P50570 |
| Sample Type: | Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids |
| Specificity: | Natural and recombinant human Dynamin-2 |
| Sub Unit: | Interacts with MYOF (By similarity). Interacts with CTTN and ACTN1 (By similarity). Interacts with SHANK1, SHANK2, SH3BP4 and NOSTRIN. Interacts with SNX9. Interacts with SNX33 (via SH3 domain). Interacts with MYO1E (via SH3 domain). Interacts with PSTPIP1. Interacts with CTNND2 (PubMed:22022388). May interact with PIK3C3 (By similarity). May be a component of a complex composed of RAB5A (in GDP-bound form), DYN2 and PIK3C3. |
| Research Area: | Cancer |
| Subcellular Location: | Cytoplasm Cytoplasm Cytoskeleton Cell junction Membrane Clathrin-coated pit Cell junction Synapse Postsynaptic cell membrane Postsynaptic density Cell junction Synapse Midbody Cell projection Phagocytic cup Cytoplasmic vesicle Phagosome membrane Peripheral membrane protein Colocalizes with CTTN at the basis of filopodia in hippocampus neuron growth zones (By similarity). Microtubule-associated. Also found in the postsynaptic density of neuronal cells. Co-localizes with PIK3C3 and RAB5A to the nascent phagosome (By similarity). |
| Storage: | Please see kit components below for exact storage details |
| Note: | For research use only |
| UniProt Protein Function: | DYN2: a cytoplasmic microtubule-associated force-producing protein involved in producing microtubule bundles and able to bind and hydrolyze GTP. Most probably involved in vesicular trafficking processes. May mediate the conventional clathrin-mediated uptake of surface receptors. Expressed in the postsynaptic density of neuronal cells. Two alternatively spliced isoforms have been described. |
| UniProt Protein Details: | Protein type:Microtubule-binding; EC 3.6.5.5; Motility/polarity/chemotaxis; Vesicle; Hydrolase Chromosomal Location of Human Ortholog: 19p13.2 Cellular Component: Golgi membrane; Golgi apparatus; postsynaptic membrane; microtubule; growth cone; focal adhesion; postsynaptic density; cytoplasm; plasma membrane; coated pit; midbody; cytosol Molecular Function:GTPase activity; protein binding; enzyme binding; GTP binding; microtubule binding; SH3 domain binding Biological Process: receptor-mediated endocytosis; positive regulation of apoptosis; positive regulation of transcription, DNA-dependent; synaptic vesicle transport; antigen processing and presentation of exogenous peptide antigen via MHC class II; transferrin transport; endocytosis; regulation of axon extension; signal transduction; nitric oxide metabolic process; regulation of transcription, DNA-dependent; receptor internalization; post-Golgi vesicle-mediated transport; regulation of nitric-oxide synthase activity; G2/M transition of mitotic cell cycle; neurite morphogenesis Disease: Lethal Congenital Contracture Syndrome 5; Myopathy, Centronuclear, 1; Charcot-marie-tooth Disease, Dominant Intermediate B |
| NCBI Summary: | Dynamins represent one of the subfamilies of GTP-binding proteins. These proteins share considerable sequence similarity over the N-terminal portion of the molecule, which contains the GTPase domain. Dynamins are associated with microtubules. They have been implicated in cell processes such as endocytosis and cell motility, and in alterations of the membrane that accompany certain activities such as bone resorption by osteoclasts. Dynamins bind many proteins that bind actin and other cytoskeletal proteins. Dynamins can also self-assemble, a process that stimulates GTPase activity. Five alternatively spliced transcripts encoding different proteins have been described. Additional alternatively spliced transcripts may exist, but their full-length nature has not been determined. [provided by RefSeq, Jun 2010] |
| UniProt Code: | P50570 |
| NCBI GenInfo Identifier: | 47117856 |
| NCBI Gene ID: | 1785 |
| NCBI Accession: | P50570.2 |
| UniProt Secondary Accession: | P50570,Q5I0Y0, Q7Z5S3, Q9UPH4, A8K1B6, E7EV30, E9PEQ4 K7ESI9, |
| UniProt Related Accession: | P50570 |
| Molecular Weight: | 870 |
| NCBI Full Name: | Dynamin-2 |
| NCBI Synonym Full Names: | dynamin 2 |
| NCBI Official Symbol: | DNM2 |
| NCBI Official Synonym Symbols: | DYN2; CMT2M; DYNII; LCCS5; CMTDI1; CMTDIB; DI-CMTB |
| NCBI Protein Information: | dynamin-2; dynamin II |
| UniProt Protein Name: | Dynamin-2 |
| Protein Family: | Dynamin |
| UniProt Gene Name: | DNM2 |
| UniProt Entry Name: | DYN2_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. |
