Mouse Heat shock 70 kDa protein 1A (Hspa1a) ELISA Kit (MOEB0646)
- SKU:
- MOEB0646
- Product Type:
- ELISA Kit
- Size:
- 96 Assays
- Uniprot:
- Q61696
- Range:
- 0.156-10 ng/mL
- ELISA Type:
- Sandwich
- Synonyms:
- DAAP-21F2.7, FLJ54328, HSP70-1B, HSP70-2, HSPA1A, heat shock 70kD protein 1B
- Reactivity:
- Mouse
Frequently bought together:
Description
Mouse Heat shock 70 kDa protein 1A (Hspa1a) ELISA Kit
The Mouse Heat Shock 70 kDa Protein 1A (Hspa1a) ELISA Kit is a powerful tool for the precise measurement of Hspa1a levels in mouse serum, plasma, and cell culture supernatants. This kit delivers outstanding accuracy and specificity, ensuring trustworthy and consistent outcomes for various research purposes.Hspa1a, also known as Heat Shock Protein 70, is a key player in cellular stress response and protein folding. It plays a critical role in maintaining cell homeostasis and protecting cells from damage caused by stressors.
Dysregulation of Hspa1a has been linked to a variety of diseases, including cancer, neurodegenerative disorders, and inflammatory conditions, making it a valuable biomarker for studying these diseases and potential therapeutic interventions.Overall, the Mouse Heat Shock 70 kDa Protein 1A (Hspa1a) ELISA Kit is a reliable tool for researchers seeking to delve deeper into the role of Hspa1a in health and disease, providing valuable insights for future studies and clinical applications.
| Product Name: | Mouse Heat shock 70 kDa protein 1A (Hspa1a) ELISA Kit |
| SKU: | MOEB0646 |
| Size: | 96T |
| Target: | Mouse Heat shock 70 kDa protein 1A (Hspa1a) |
| Synonyms: | Heat shock 70 kDa protein 3, Hsp68, HSP70.3, Hsp70-3, Hsp70A1 |
| Assay Type: | Sandwich |
| Detection Method: | ELISA |
| Reactivity: | Mouse |
| Detection Range: | 0.156-10ng/mL |
| Sensitivity: | 0.078ng/mL |
| Intra CV: | 4.2% | ||||||||||||||||||||
| Inter CV: | 6.4% | ||||||||||||||||||||
| Linearity: |
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| Recovery: |
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| Function: | Molecular chaperone implicated in a wide variety of cellular processes, including protection of the proteome from stress, folding and transport of newly synthesized polypeptides, activation of proteolysis of misfolded proteins and the formation and dissociation of protein complexes. Plays a pivotal role in the protein quality control system, ensuring the correct folding of proteins, the re-folding of misfolded proteins and controlling the targeting of proteins for subsequent degradation. This is achieved through cycles of ATP binding, ATP hydrolysis and ADP release, mediated by co-chaperones. The co-chaperones have been shown to not only regulate different steps of the ATPase cycle, but they also have an individual specificity such that one co-chaperone may promote folding of a substrate while another may promote degradation. The affinity for polypeptides is regulated by its nucleotide bound state. In the ATP-bound form, it has a low affinity for substrate proteins. However, upon hydrolysis of the ATP to ADP, it undergoes a conformational change that increases its affinity for substrate proteins. It goes through repeated cycles of ATP hydrolysis and nucleotide exchange, which permits cycles of substrate binding and release. The co-chaperones are of three types: J-domain co-chaperones such as HSP40s (stimulate ATPase hydrolysis by HSP70), the nucleotide exchange factors (NEF) such as BAG1/2/3 (facilitate conversion of HSP70 from the ADP-bound to the ATP-bound state thereby promoting substrate release), and the TPR domain chaperones such as HOPX and STUB1. Maintains protein homeostasis during cellular stress through two opposing mechanisms: protein refolding and degradation. Its acetylation/deacetylation state determines whether it functions in protein refolding or protein degradation by controlling the competitive binding of co-chaperones HOPX and STUB1. During the early stress response, the acetylated form binds to HOPX which assists in chaperone-mediated protein refolding, thereafter, it is deacetylated and binds to ubiquitin ligase STUB1 that promotes ubiquitin-mediated protein degradation. Regulates centrosome integrity during mitosis, and is required for the maintenance of a functional mitotic centrosome that supports the assembly of a bipolar mitotic spindle. Enhances STUB1-mediated SMAD3 ubiquitination and degradation and facilitates STUB1-mediated inhibition of TGF-beta signaling. Essential for STUB1-mediated ubiquitination and degradation of FOXP3 in regulatory T-cells (Treg) during inflammation. Negatively regulates heat shock-induced HSF1 transcriptional activity during the attenuation and recovery phase period of the heat shock response. |
| Uniprot: | Q61696 |
| Sample Type: | Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids |
| Specificity: | Natural and recombinant mouse Heat shock 70 kDa protein 1A |
| Sub Unit: | Component of the CatSper complex (By similarity). Identified in a IGF2BP1-dependent mRNP granule complex containing untranslated mRNAs (By similarity). Interacts with CHCHD3, DNAJC7, IRAK1BP1, PPP5C and TSC2 (By similarity). Interacts with TERT; the interaction occurs in the absence of the RNA component, TERC, and dissociates once the TERT complex has formed (By similarity). Interacts with METTL21A (By similarity). Interacts with DNAAF2 (PubMed:19052621). Interacts with TRIM5 (via B30.2/SPRY domain) (By similarity). Interacts with PARK2 (By similarity). Interacts with FOXP3 (PubMed:23973223). Interacts with NOD2; the interaction enhances NOD2 stability (By similarity). Interacts with DNAJC9 (via J domain) (By similarity). Interacts with ATF5; the interaction protects ATF5 from degradation via proteasome-dependent and caspase-dependent processes (By similarity). Interacts with RNF207 (via the C-terminus); this interaction additively increases KCNH2 expression (By similarity). Interacts with HSF1 (via transactivation domain); this interaction results in the inhibition of heat shock- and HSF1-induced transcriptional activity during the attenuation and recovery phase period of the heat shock response. Interacts with NAA10, HSP40, HSP90 and HDAC4. The acetylated form and the non-acetylated form interact with HOPX and STUB1 respectively. Interacts with NEDD1 and SMAD3. Interacts (via NBD) with BAG1, BAG2, BAG3 and HSPH1/HSP105. |
| Research Area: | Neurosciences |
| Subcellular Location: | Cytoplasm Nucleus Cytoplasm Cytoskeleton Microtubule organizing center Centrosome Localized in cytoplasmic mRNP granules containing untranslated mRNAs. |
| Storage: | Please see kit components below for exact storage details |
| Note: | For research use only |
| UniProt Protein Function: | HSP70: a critical chaperone protein that has a high affinity for unfolded polypeptide chains. It binds extended peptide segments with a net hydrophobic character exposed by polypeptides during translation and membrane translocation, or following stress-induced damage. In cooperation with other chaperones, hsp70 stabilizes preexistent proteins against aggregation and mediates the folding of newly translated polypeptides in the cytosol as well as within organelles. Mitochondrial HSP70 is crucial to the import process: mutant forms of HSP70 fail to import precursor proteins. Is anti-apoptotic in sympathetic neurones and mediates this effect primarily by suppressing c-Jun transcriptional signalling. Interacts with tau protein and mediates proper folding of tau. Can promote the degradation of tau protein. Triptolide, a potential therapeutic agent for progression/metastasis of pancreatic cancer, causes pancreatic cancer cell death by induction of apoptosis, an effect mediated by the inhibition of HSP70.Protein type: Heat shock protein; Chaperone; Motility/polarity/chemotaxisCellular Component: signalosome; focal adhesion; protein complex; mitochondrion; basolateral plasma membrane; inclusion body; cytosol; ribonucleoprotein complex; centriole; perinuclear region of cytoplasm; apical plasma membrane; cytoplasm; nuclear speck; nucleus; vesicle; ubiquitin ligase complexMolecular Function: NF-kappaB binding; enzyme binding; protease binding; G-protein-coupled receptor binding; ubiquitin protein ligase binding; heat shock protein binding; unfolded protein binding; ATPase activity; double-stranded RNA binding; protein N-terminus binding; ATPase activity, coupled; ATP bindingBiological Process: protein folding; response to heat; DNA repair; telomere maintenance |
| UniProt Protein Details: | |
| NCBI Summary: | |
| UniProt Code: | Q61696 |
| NCBI GenInfo Identifier: | 124339829 |
| NCBI Gene ID: | 193740 |
| NCBI Accession: | NP_034609.2 |
| UniProt Secondary Accession: | Q61696,Q61697, Q7TQD8, Q9QWJ5 |
| UniProt Related Accession: | Q61696 |
| Molecular Weight: | Predicted Molecular Mass: 60.8kDaAccurate Molecular Mass: 60kDa as determined by SDS-PAGE reducing conditions. |
| NCBI Full Name: | heat shock 70 kDa protein 1A |
| NCBI Synonym Full Names: | heat shock protein 1A |
| NCBI Official Symbol: | Hspa1a |
| NCBI Official Synonym Symbols: | Hsp72; hsp68; Hsp70-3; Hsp70.3; hsp70A1 |
| NCBI Protein Information: | heat shock 70 kDa protein 1A; 68 kDa heat shock protein; heat shock 70 kDa protein 3; heat shock 70kDa protein 1A; heat shock protein, 70 kDa 3; inducible heat shock protein 70 |
| UniProt Protein Name: | Heat shock 70 kDa protein 1A |
| UniProt Synonym Protein Names: | Heat shock 70 kDa protein 3; HSP70.3; Hsp68 |
| Protein Family: | Heat shock 70 kDa protein |
| UniProt Gene Name: | Hspa1a |
| UniProt Entry Name: | HS71A_MOUSE |
| 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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