Mouse Cell Signalling ELISA Kits 4
Mouse TSC22 domain family protein 1 (Tsc22d1) ELISA Kit
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- SKU:
- MOEB1580
- Product Type:
- ELISA Kit
- Size:
- 96 Assays
- Uniprot:
- P62500
- ELISA Type:
- Sandwich
- Reactivity:
- Mouse
Description
| Product Name: | Mouse TSC22 domain family protein 1 (Tsc22d1) ELISA Kit |
| Product Code: | MOEB1580 |
| Alias: | TSC22 domain family protein 1, Regulatory protein TSC-22, TGFB-stimulated clone 22 homolog, TSC22-related inducible leucine zipper 1b, Transforming growth factor beta-1-induced transcript 4 protein, Tsc22d1, Kiaa1994, Tgfb1i4, Tilz1b, Tsc22 |
| Uniprot: | P62500 |
| Reactivity: | Mouse |
| 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: | NUP62: Essential component of the nuclear pore complex. The N- terminal is probably involved in nucleocytoplasmic transport. The C-terminal is probably involved in protein-protein interaction via coiled-coil formation and may function in anchorage of p62 to the pore complex. Defects in NUP62 are the cause of infantile striatonigral degeneration (SNDI); also known as infantile bilateral striatal necrosis (IBSN) or familial striatal degeneration. SNDI is a neurological disorder characterized by symmetrical degeneration of the caudate nucleus, putamen and occasionally the globus pallidus, with little involvement of the rest of the brain. The clinical features include developmental regression, choreoathetosis, dystonia, spasticity, dysphagia, failure to thrive, nystagmus, optic atrophy and mental retardation. Belongs to the nucleoporin NSP1/NUP62 family.Protein type: Adaptor/scaffold; NucleoporinChromosomal Location of Human Ortholog: 19q13.33Cellular Component: annulate lamellae; cytoplasm; intracellular membrane-bound organelle; nuclear envelope; nuclear membrane; nuclear pore; nucleocytoplasmic shuttling complex; pore complex; ribonucleoprotein complex; spindle poleMolecular Function: chromatin binding; kinesin binding; nucleocytoplasmic transporter activity; protein binding; receptor signaling complex scaffold activity; SH2 domain binding; structural constituent of nuclear pore; thyroid hormone receptor binding; ubiquitin bindingBiological Process: carbohydrate metabolic process; cell death; cell surface receptor linked signal transduction; cellular protein metabolic process; cytokine and chemokine mediated signaling pathway; gene expression; glucose transport; hexose transport; hormone-mediated signaling; mitosis; mitotic cell cycle; mitotic nuclear envelope disassembly; modification by virus of host cellular process; mRNA transport; negative regulation of apoptosis; negative regulation of cell proliferation; negative regulation of programmed cell death; positive regulation of epidermal growth factor receptor signaling pathway; positive regulation of I-kappaB kinase/NF-kappaB cascade; positive regulation of transcription, DNA-dependent; post-translational protein modification; protein import into nucleus; protein sumoylation; regulation of protein import into nucleus; regulation of Ras protein signal transduction; regulation of signal transduction; RNA-mediated gene silencing; spermatogenesis; transcription, DNA-dependent; transmembrane transport; tRNA processing; viral infectious cycle; viral reproduction; viral transcription; virus-host interactionDisease: Striatonigral Degeneration, Infantile |
| UniProt Protein Details: | |
| NCBI Summary: | The nuclear pore complex is a massive structure that extends across the nuclear envelope, forming a gateway that regulates the flow of macromolecules between the nucleus and the cytoplasm. Nucleoporins are the main components of the nuclear pore complex in eukaryotic cells. The protein encoded by this gene is a member of the FG-repeat containing nucleoporins and is localized to the nuclear pore central plug. This protein associates with the importin alpha/beta complex which is involved in the import of proteins containing nuclear localization signals. Multiple transcript variants of this gene encode a single protein isoform. [provided by RefSeq, Jul 2008] |
| UniProt Code: | P62500 |
| NCBI GenInfo Identifier: | 15928767 |
| NCBI Gene ID: | 23636 |
| NCBI Accession: | AAH14842 |
| UniProt Secondary Accession: | P62500,Q503A4, Q6GTM2, Q96C43, Q9NSL1, B3KWU5 |
| UniProt Related Accession: | P62500,P37198 |
| Molecular Weight: | 53,255 Da |
| NCBI Full Name: | Nucleoporin 62kDa |
| NCBI Synonym Full Names: | nucleoporin 62kDa |
| NCBI Official Symbol: | NUP62 |
| NCBI Official Synonym Symbols: | p62; IBSN; SNDI |
| NCBI Protein Information: | nuclear pore glycoprotein p62 |
| UniProt Protein Name: | Nuclear pore glycoprotein p62 |
| UniProt Synonym Protein Names: | 62 kDa nucleoporin; Nucleoporin Nup62 |
| Protein Family: | Nuclear pore glycoprotein |
| UniProt Gene Name: | NUP62 |
| UniProt Entry Name: | NUP62_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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