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Mouse Heat shock cognate 71 kDa protein (Hspa8) ELISA Kit (MOEB0859)

SKU:
MOEB0859
Product Type:
ELISA Kit
Size:
96 Assays
Uniprot:
P63017
ELISA Type:
Sandwich
Reactivity:
Mouse
€649

Description

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Mouse Heat shock cognate 71 kDa protein (Hspa8) ELISA Kit

The Mouse Heat Shock Cognate 71 kDa Protein (HSP A8) ELISA Kit is specifically designed for the accurate detection of HSP A8 levels in mouse serum, plasma, and cell culture supernatants. This kit offers high sensitivity and specificity, ensuring reliable and reproducible results for a variety of research applications.HSP A8, also known as heat shock protein 70 (HSP70), is a chaperone protein involved in cellular stress response and protein folding. It plays a critical role in maintaining cellular homeostasis and protecting cells from environmental stressors.

Dysregulation of HSP A8 has been linked to various diseases, including cancer, neurodegenerative disorders, and cardiovascular diseases, making it a valuable biomarker for studying these conditions and developing potential therapies.Overall, the Mouse Heat Shock Cognate 71 kDa Protein (HSP A8) ELISA Kit is a reliable tool for researchers seeking to investigate the role of HSP A8 in health and disease.

Product Name:Mouse Heat shock cognate 71 kDa protein (Hspa8) ELISA Kit
SKU:MOEB0859
Size:96T
Target:Mouse Heat shock cognate 71 kDa protein (Hspa8)
Synonyms:Heat shock 70 kDa protein 8, Hsc70, Hsc73
Assay Type:Sandwich
Detection Method:ELISA
Reactivity:Mouse
Detection Range:0.312-20ng/mL
Sensitivity:0.167ng/mL
Intra CV:Provided with the Kit
Inter CV:Provided with the Kit
Linearity:Provided with the Kit
Recovery:Provided with the Kit
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 of HSP70, 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 of HSP70 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. HSP70 goes through repeated cycles of ATP hydrolysis and nucleotide exchange, which permits cycles of substrate binding and release. The HSP70-associated co-chaperones are of three types: J-domain co-chaperones 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. Acts as a repressor of transcriptional activation. Inhibits the transcriptional coactivator activity of CITED1 on Smad-mediated transcription. Component of the PRP19-CDC5L complex that forms an integral part of the spliceosome and is required for activating pre-mRNA splicing. May have a scaffolding role in the spliceosome assembly as it contacts all other components of the core complex. Binds bacterial lipopolysaccharide (LPS) and mediates LPS-induced inflammatory response, including TNF secretion. Participates in the ER-associated degradation (ERAD) quality control pathway in conjunction with J domain-containing co-chaperones and the E3 ligase STUB1.
Uniprot:P63017
Sample Type:Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids
Specificity:Natural and recombinant mouse Heat shock cognate 71 kDa protein
Sub Unit:Identified in a IGF2BP1-dependent mRNP granule complex containing untranslated mRNAs. Interacts with PACRG. Interacts with DNAJC7. Interacts with DNAJB12 (via J domain). Interacts with DNAJB14 (via J domain). Interacts (via C-terminus) with the E3 ligase STUB1 forming a 210 kDa complex of one STUB1 and two HSPA8 molecules. Interacts with CITED1 (via N-terminus); the interaction suppresses the association of CITED1 to p300/CBP and Smad-mediated transcription transactivation. Component of the PRP19-CDC5L splicing complex composed of a core complex comprising a homotetramer of PRPF19, CDC5L, PLRG1 and BCAS2, and at least three less stably associated proteins CTNNBL1, CWC15 and HSPA8. Interacts with IRAK1BP1 and HSPH1/HSP105 (PubMed:9675148, PubMed:15292236, PubMed:17233114). Interacts with TRIM5. Part of a complex composed at least of ASCL2, EMSY, HCFC1, HSPA8, CCAR2, MATR3, MKI67, RBBP5, TUBB2A, WDR5 and ZNF335; this complex may have a histone H3-specific methyltransferase activity. Following LPS binding, may form a complex with CXCR4, GDF5 and HSP90AA1. Interacts with PARK2. Interacts with FOXP3. Interacts with DNAJC9 (via J domain). Interacts with MLLT11. Interacts with RNF207. Interacts with DNAJC21. Interacts with DNAJB2. Interacts with TTC1 (via TPR repeats). Interacts with SGTA (via TPR repeats). Interacts with HSF1 (via transactivation domain). Interacts with HOPX, STUB1, HSP40, HSP90, BAG2 and BAG3.
Research Area:Epigenetics
Subcellular Location:Cytoplasm Melanosome Nucleus Nucleolus Cell membrane Localized in cytoplasmic mRNP granules containing untranslated mRNAs. Translocates rapidly from the cytoplasm to the nuclei, and especially to the nucleoli, upon heat shock (By similarity).
Storage:Please see kit components below for exact storage details
Note:For research use only
UniProt Protein Function:HSC70: Acts as a repressor of transcriptional activation. Inhibits the transcriptional coactivator activity of CITED1 on Smad-mediated transcription. Chaperone. Isoform 2 may function as an endogenous inhibitory regulator of HSC70 by competing the co- chaperones. Interacts with HSPH1/HSP105. Interacts with IRAK1BP1. Identified in a mRNP granule complex, at least composed of ACTB, ACTN4, DHX9, ERG, HNRNPA1, HNRNPA2B1, HNRNPAB, HNRNPD, HNRNPL, HNRNPR, HNRNPU, HSPA1, HSPA8, IGF2BP1, ILF2, ILF3, NCBP1, NCL, PABPC1, PABPC4, PABPN1, RPLP0, RPS3, RPS3A, RPS4X, RPS8, RPS9, SYNCRIP, TROVE2, YBX1 and untranslated mRNAs. Interacts with PACRG and TSC2. Interacts with BAG1. Interacts with SV40 VP1. Interacts with DNAJC7. Interacts with HERC5. Interacts with CITED1 (via N-terminus); the interaction suppresses the association of CITED1 to p300/CBP and Smad-mediated transcription transactivation. Constitutively synthesized. Ubiquitous. Belongs to the heat shock protein 70 family. 2 isoforms of the human protein are produced by alternative splicing.Protein type: RNA-binding; Chaperone; Heat shock protein; NucleolusChromosomal Location of Human Ortholog: 11q24.1Cellular Component: nucleoplasm; spliceosome; extracellular space; focal adhesion; membrane; melanosome; nucleolus; plasma membrane; intracellular; ribonucleoprotein complex; nucleus; cytosol; ubiquitin ligase complexMolecular Function: protein binding; enzyme binding; G-protein-coupled receptor binding; heat shock protein binding; ubiquitin protein ligase binding; ATPase activity; unfolded protein binding; ATPase activity, coupled; ATP bindingBiological Process: axon guidance; viral reproduction; chaperone cofactor-dependent protein folding; protein folding; transcription, DNA-dependent; regulation of cell cycle; neurotransmitter secretion; RNA splicing; response to unfolded protein; nuclear mRNA splicing, via spliceosome; positive regulation of nuclear mRNA splicing, via spliceosome; synaptic transmission; gene expression; protein refolding; post-Golgi vesicle-mediated transport; negative regulation of transcription, DNA-dependent
UniProt Protein Details:
NCBI Summary:This gene encodes a member of the heat shock protein 70 family, which contains both heat-inducible and constitutively expressed members. This protein belongs to the latter group, which are also referred to as heat-shock cognate proteins. It functions as a chaperone, and binds to nascent polypeptides to facilitate correct folding. It also functions as an ATPase in the disassembly of clathrin-coated vesicles during transport of membrane components through the cell. Alternatively spliced transcript variants encoding different isoforms have been found for this gene. [provided by RefSeq, Aug 2011]
UniProt Code:P63017
NCBI GenInfo Identifier:5729877
NCBI Gene ID:3312
NCBI Accession:NP_006588.1
UniProt Secondary Accession:P63017,P63017, P63018
UniProt Related Accession:P63017,P11142
Molecular Weight:
NCBI Full Name:heat shock cognate 71 kDa protein isoform 1
NCBI Synonym Full Names:heat shock protein family A (Hsp70) member 8
NCBI Official Symbol:HSPA8
NCBI Official Synonym Symbols:LAP1; HSC54; HSC70; HSC71; HSP71; HSP73; LAP-1; NIP71; HEL-33; HSPA10; HEL-S-72p
NCBI Protein Information:heat shock cognate 71 kDa protein
UniProt Protein Name:Heat shock cognate 71 kDa protein
UniProt Synonym Protein Names:Heat shock 70 kDa protein 8
Protein Family:Hsc70-interacting protein
UniProt Gene Name:HSPA8
UniProt Entry Name:HSP7C_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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