Human Heat Shock Protein 70,HSP-70 ELISA Kit

Instructions
Code CSB-E08297h
Size 96T,5×96T,10×96T
Trial Size 24T ELISA kits trial application
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Product Details

Description

The Human Heat Shock Protein 70 (HSP70) ELISA Kit allows for the in vitro quantitative determination of HSP70 concentrations in serum, plasma, or tissue homogenates. This assay kit was designed and optimized for neuroscience research use in humans. It has undergone rigorous quality control in multiple parameters, including sensitivity, specificity, precision, linearity, recovery, and inter-batch difference. Refer to the product instructions for more details.

This assay employs the quantitative sandwich enzyme immunoassay technique, in which HSP70 in the samples or standards are sandwiched between pre-coated HSP70 antibody and Biotin-conjugated HSP70 antibody. HRP-avidin is then added into the wells. Following a wash to remove any unbound reagent, the TMB substrate solution is added to the wells and color develops in proportion to the amount of HSP70 bound in the initial step. The color development is stopped upon adding the stop solution, and the intensity of the color is measured at 450 nm via a microplate reader. The levels of HSP70 in the samples can be determined by referring to the O.D. (optical density) of the samples to the standard curve.

HSP70 is generated in response to stress or constitutively as the major chaperone contributing to maintaining protein homeostasis in both eukaryotes and prokaryotes. HSP70 binds to client proteins in the early stages of protein folding, which controls the availability of such regions, promoting the formation of the proper protein fold, but inhibiting aggregate formation. Upon detection of incorrect protein folding, HSP70 associates with additional co-chaperones to facilitate degradation of the misfolded protein. It also enhances bacterial survival in hostile environments.

Target Name heat shock 70kDa protein 1B
Alternative Names HSPA1B ELISA Kit; HSP72 ELISA Kit; Heat shock 70 kDa protein 1B ELISA Kit; Heat shock 70 kDa protein 2 ELISA Kit; HSP70-2 ELISA Kit; HSP70.2 ELISA Kit
Abbreviation HSPA1B
Uniprot No. P0DMV9
Species Homo sapiens (Human)
Sample Types serum, plasma, tissue homogenates
Detection Range 1.56 ng/mL-100 ng/mL
Sensitivity 0.39 ng/mL
Assay Time 1-5h
Sample Volume 50-100ul
Detection Wavelength 450 nm
Research Area Neuroscience
Assay Principle quantitative
Measurement Sandwich
Precision
Intra-assay Precision (Precision within an assay): CV%<8%
Three samples of known concentration were tested twenty times on one plate to assess.
Inter-assay Precision (Precision between assays): CV%<10%
Three samples of known concentration were tested in twenty assays to assess.
Linearity
To assess the linearity of the assay, samples were spiked with high concentrations of human HSP-70 in various matrices and diluted with the Sample Diluent to produce samples with values within the dynamic range of the assay.
 SampleSerum(n=4)
1:1Average %96
Range %85-100
1:2Average %87
Range %82-93
1:4Average %98
Range %95-100
1:8Average %92
Range %88-94
Recovery
The recovery of human HSP-70 spiked to levels throughout the range of the assay in various matrices was evaluated. Samples were diluted prior to assay as directed in the Sample Preparation section.
Sample TypeAverage % RecoveryRange
Serum (n=5) 10095-106
EDTA plasma (n=4)9287-96
Typical Data
These standard curves are provided for demonstration only. A standard curve should be generated for each set of samples assayed.
ng/mlOD1OD2AverageCorrected
1002.588 2.521 2.555 2.445
501.745 1.676 1.711 1.601
251.133 1.112 1.123 1.013
12.50.667 0.621 0.644 0.534
6.250.401 0.399 0.400 0.290
3.120.238 0.233 0.236 0.126
1.560.167 0.159 0.163 0.053
00.112 0.108 0.110  
Materials provided
  • A micro ELISA plate ---The 96-well plate has been pre-coated with an anti-human HSP70 antibody. This dismountable microplate can be divided into 12 x 8 strip plates.
  • Two vials lyophilized standard ---Dilute a bottle of the standard at dilution series, read the OD values, and then draw a standard curve.
  • One vial Biotin-labeled HSP70 antibody (100 x concentrate) (120 μl/bottle) ---Act as the detection antibody.
  • One vial HRP-avidin (100 x concentrate) (120 μl/bottle) ---Bind to the detection antibody and react with the TMB substrate to make the solution chromogenic.
  • One vial Biotin-antibodyDiluent (15 ml/bottle) ---Dilute the Biotin-antibody.
  • One vial HRP-avidin Diluent (15 ml/bottle) ---Dilute the HRP-avidin solution.
  • One vial Sample Diluent (50 ml/bottle)---Dilute the sample to an appropriate concentration.
  • One vial Wash Buffer (25 x concentrate) (20 ml/bottle) ---Wash away unbound or free substances.
  • One vial TMB Substrate (10 ml/bottle) ---Act as the chromogenic agent. TMB interacts with HRP, eliciting the solution turns blue.
  • One vial Stop Solution (10 ml/bottle) ---Stop the color reaction. The solution color immediately turns from blue to yellow.
  • Four Adhesive Strips (For 96 wells) --- Cover the microplate when incubation.
  • An instruction manual
Materials not provided
  • A microplate reader capable of measuring absorbance at 450 nm, with the correction wavelength set at 540 nm or 570 nm.
  • An incubator can provide stable incubation conditions up to 37°C±5°C.
  • Centrifuge
  • Vortex
  • Squirt bottle, manifold dispenser, or automated microplate washer
  • Absorbent paper for blotting the microtiter plate
  • 50-300ul multi-channel micropipette
  • Pipette tips
  • Single-channel micropipette with different ranges
  • 100ml and 500ml graduated cylinders
  • Deionized or distilled water
  • Timer
  • Test tubes for dilution
Troubleshooting
and FAQs
ELISA kit FAQs
Storage Store at 2-8°C. Please refer to protocol.
Lead Time 3-5 working days

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Target Data

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
Subcellular Location Cytoplasm, Cytoplasm, cytoskeleton, microtubule organizing center, centrosome
Protein Families Heat shock protein 70 family
Tissue Specificity HSPA1B is testis-specific.
Database Links

HGNC: 5233

OMIM: 140550

KEGG: hsa:3303

UniGene: Hs.274402

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