Description
Human Tyrosine-protein kinase ZAP-70 (ZAP70) ELISA Kit
The Human Tyrosine Protein Kinase ZAP-70 (ZAP70) ELISA Kit is a advanced tool for the precise measurement of ZAP70 levels in human samples such as serum, plasma, and cell culture supernatants. This kit is engineered with exceptional sensitivity and specificity, ensuring dependable and consistent results for various research applications.ZAP70 is a critical enzyme involved in the regulation of immune cell signaling, particularly in T cells. Dysregulation of ZAP70 has been linked to autoimmune diseases and certain types of cancers, highlighting its importance as a biomarker for studying these conditions and developing targeted therapies.
With its cutting-edge technology and reliable performance, the Human Tyrosine Protein Kinase ZAP-70 (ZAP70) ELISA Kit is an invaluable asset for researchers looking to unravel the complexities of immune cell signaling pathways and explore novel treatment strategies for various diseases.
| Product Name: | Human Tyrosine-protein kinase ZAP-70 (ZAP70) ELISA Kit |
| SKU: | HUEB2540 |
| Size: | 96T |
| Target: | Human Tyrosine-protein kinase ZAP-70 (ZAP70) |
| Synonyms: | 70 kDa zeta-chain associated protein, Syk-related tyrosine kinase, SRK |
| Assay Type: | Sandwich |
| Detection Method: | ELISA |
| Reactivity: | Human |
| Detection Range: | 0.312-20ng/mL |
| Sensitivity: | 0.2ng/mL |
| Intra CV: | 5.7% | ||||||||||||||||||||
| Inter CV: | 9.8% | ||||||||||||||||||||
| Linearity: |
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| Recovery: |
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| Function: | Tyrosine kinase that plays an essential role in regulation of the adaptive immune response. Regulates motility, adhesion and cytokine expression of mature T-cells, as well as thymocyte development. Contributes also to the development and activation of primary B-lymphocytes. When antigen presenting cells (APC) activate T-cell receptor (TCR), a serie of phosphorylations lead to the recruitment of ZAP70 to the doubly phosphorylated TCR component CD247/CD3Z through ITAM motif at the plasma membrane. This recruitment serves to localization to the stimulated TCR and to relieve its autoinhibited conformation. Release of ZAP70 active conformation is further stabilized by phosphorylation mediated by LCK. Subsequently, ZAP70 phosphorylates at least 2 essential adapter proteins: LAT and LCP2. In turn, a large number of signaling molecules are recruited and ultimately lead to lymphokine production, T-cell proliferation and differentiation. Furthermore, ZAP70 controls cytoskeleton modifications, adhesion and mobility of T-lymphocytes, thus ensuring correct delivery of effectors to the APC. ZAP70 is also required for TCR-CD247/CD3Z internalization and degradation through interaction with the E3 ubiquitin-protein ligase CBL and adapter proteins SLA and SLA2. Thus, ZAP70 regulates both T-cell activation switch on and switch off by modulating TCR expression at the T-cell surface. During thymocyte development, ZAP70 promotes survival and cell-cycle progression of developing thymocytes before positive selection (when cells are still CD4/CD8 double negative). Additionally, ZAP70-dependent signaling pathway may also contribute to primary B-cells formation and activation through B-cell receptor (BCR). |
| Uniprot: | P43403 |
| Sample Type: | Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids |
| Specificity: | Natural and recombinant human Tyrosine-protein kinase ZAP-70 |
| Sub Unit: | Interacts with CD247/CD3Z; this interaction docks ZAP70 at the stimulated TCR (PubMed:1423621, PubMed:7659156, PubMed:26783323). Interacts with NFAM1 (PubMed:15143214). Interacts with adapter protein SLA; this interaction negatively regulates T-cell receptor signaling (PubMed:10449770). Interacts with FCRL3 (PubMed:12051764). Interacts with VAV1 (PubMed:9151714). Interacts with CBL; this interaction promotes ubiquitination, internalization and subsequent degradation of CD247/CD3Z (PubMed:10449770, PubMed:10078535). Identified in a complex with CBL and UBE2L3 (PubMed:10966114). Interacts with SHB (PubMed:12084069). Interacts with adapter protein SLA2; this interaction negatively regulates T-cell receptor signaling. Interacts with CBLB. Interacts (via SH2 domains) with RHOH; this interaction regulates ZAP70 subcellular localization. Interacts with DEF6 (By similarity). Interacts (ubiquitinated form) with OTUD7B and UBASH3B (PubMed:26903241). |
| Research Area: | Cancer |
| Subcellular Location: | Cytoplasm Cell membrane Peripheral membrane protein In quiescent T-lymphocytes, it is cytoplasmic. Upon TCR activation, it is recruited at the plasma membrane by interacting with CD247/CD3Z. Colocalizes together with RHOH in the immunological synapse. RHOH is required for its proper localization to the cell membrane and cytoskeleton fractions in the thymocytes (By similarity). |
| Storage: | Please see kit components below for exact storage details |
| Note: | For research use only |
| UniProt Protein Function: | ZAP70: a tyrosine kinase of the Syk family. Associates with the T-cell antigen receptor zeta-chain after TCR stimulation. Phosphorylated by Src-family kinases following antigen receptor activation. Plays a role in lymphocyte activation. |
| UniProt Protein Details: | Protein type:Kinase, protein; Protein kinase, TK; EC 2.7.10.2; Protein kinase, tyrosine (non-receptor); TK group; Syk family Chromosomal Location of Human Ortholog: 2q12 Cellular Component: cytoplasm; cytosol; extrinsic to internal side of plasma membrane; immunological synapse; intercellular junction; lipid raft; plasma membrane; T cell receptor complex Molecular Function:ATP binding; non-membrane spanning protein tyrosine kinase activity; phosphotyrosine binding; protein binding; protein-tyrosine kinase activity; receptor binding Biological Process: adaptive immune response; B cell activation; B cell receptor signaling pathway; beta selection; immune response; innate immune response; macrophage activation during immune response; negative thymic T cell selection; neutrophil activation during immune response; peptidyl-tyrosine phosphorylation; positive regulation of alpha-beta T cell differentiation; positive regulation of alpha-beta T cell proliferation; positive regulation of B cell differentiation; positive regulation of calcium-mediated signaling; positive regulation of cell adhesion mediated by integrin; positive regulation of mast cell degranulation; positive regulation of T cell differentiation; positive thymic T cell selection; protein amino acid phosphorylation; T cell activation; T cell differentiation; T cell receptor signaling pathway; transmembrane receptor protein tyrosine kinase signaling pathway Disease: Selective T-cell Defect |
| NCBI Summary: | This gene encodes an enzyme belonging to the protein tyrosine kinase family, and it plays a role in T-cell development and lymphocyte activation. This enzyme, which is phosphorylated on tyrosine residues upon T-cell antigen receptor (TCR) stimulation, functions in the initial step of TCR-mediated signal transduction in combination with the Src family kinases, Lck and Fyn. This enzyme is also essential for thymocyte development. Mutations in this gene cause selective T-cell defect, a severe combined immunodeficiency disease characterized by a selective absence of CD8-positive T-cells. Two transcript variants that encode different isoforms have been found for this gene. [provided by RefSeq, Jul 2008] |
| UniProt Code: | P43403 |
| NCBI GenInfo Identifier: | 1177044 |
| NCBI Gene ID: | 7535 |
| NCBI Accession: | P43403.1 |
| UniProt Secondary Accession: | P43403,Q6PIA4, Q8IXD6, Q9UBS6, A6NFP4, |
| UniProt Related Accession: | P43403 |
| Molecular Weight: | 55,873 Da |
| NCBI Full Name: | Tyrosine-protein kinase ZAP-70 |
| NCBI Synonym Full Names: | zeta chain of T cell receptor associated protein kinase 70kDa |
| NCBI Official Symbol: | ZAP70 |
| NCBI Official Synonym Symbols: | SRK; STD; TZK; STCD; ZAP-70 |
| NCBI Protein Information: | tyrosine-protein kinase ZAP-70 |
| UniProt Protein Name: | Tyrosine-protein kinase ZAP-70 |
| UniProt Synonym Protein Names: | 70 kDa zeta-chain associated protein; Syk-related tyrosine kinase |
| Protein Family: | Tyrosine-protein kinase |
| UniProt Gene Name: | ZAP70 |
| UniProt Entry Name: | ZAP70_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. |
