SKU EK7115
Size 96wells/kit, with removable strips.
Reactivity Alfalfa, Flax, Human, Maize, Plant
Sample Type Cell lysates, Plant extract, Plasma, Serum, Tissue
Sample Volume 100ul per well
Sensitivity 0.18 ng/ml
Assay Range 1.563 - 100 ng/ml

Introduction

Boster's ELISA Kit is for the detection of cytoplasmic Plant Hsp70 from plant extracts. But this kit is also compatible with human tissue extracts and lysates and serum and plasma. Each kit contains sufficient components to quantitate the Hsp70 concentration in up to 40 samples, tested in duplicate.

Overview

Product Name HSP70 ELISA Kit (PLANT)
SKU/Catalog Number EK7115
Storage & Handling Store at 4°C.
Size 96wells/kit, with removable strips.
Description Colorimetric detection used to quantitate HSP70 in plants. 96wells/kit, with removable strips.
Cite This Product HSP70 ELISA Kit (PLANT) (Boster Biological Technology, Pleasanton CA, USA, Catalog # EK7115)
Sample Type Cell lysates, Plant extract, Plasma, Serum, Tissue
Sensitivity 0.18 ng/ml
Assay Range 1.563 - 100 ng/ml
Reactivity Alfalfa, Flax, Human, Maize, Plant
Cross Reactivity There is no detectable cross-reactivity.

Assay Details

Kit Components

DescriptionQuantity
Anti-Hsp70 Immunoassay Plate1 Plate
5X Hsp70 Extraction Reagent1 vial/10 ml
Recombinant Hsp70 (Alfalfa) Standard2 vials
Standard and Sample Diluent1 vial/ 50 ml
10X Wash Buffer Concentrate1 vial/100 ml
Anti-Hsp70 Biotinylated Antibody Concentrate1 vial/150 µl
Anti-Hsp70 Biotinylated Antibody Diluent1 vial/ 13 ml
Streptavidin: HRP Concentrate1 vial/150 µl
Streptavidin: HRP Diluent1 vial/ 13 ml
TMB Substrate1 vial/ 13 ml
Stop Solution1 vial/ 13 ml

*Why there is no wash buffer? Our Avidin-Biotin-Peroxidase Diluent contains the detergent (TWEEN) normally present in other companies' ELISA kits. This saves you the step of having to wash with the special wash buffer and achieve similar or better signal to noise ratio. The wash can use regular wash buffers (PBS, TBS etc.) commonly found in labs.

Materials Required But Not Provided

  • Microplate reader in standard size.
  • Automated plate washer.
  • Adjustable pipettes and pipette tips. Multichannel pipettes are recommended in the condition of large amount of samples in the detection.
  • Clean tubes and Eppendorf tubes.
  • Washing buffer (neutral PBS or TBS).
  • Preparation of 0.01M TBS: Add 1.2g Tris, 8.5g NaCl; 450μl of purified acetic acid or 700μl of concentrated hydrochloric acid to 1000ml H2
  • Preparation of 0.01 M PBS: Add 8.5g sodium chloride, 1.4g Na O and adjust pH to 7.2-7.

*The typical data is obtained from Boster's internal QC result and for reference only. It may differ from the lab test results of the end users. It is more important that the user's lab test results reflect the same linearity demonstrated in the typical data than achieving exactly the same O.D. values.

Target Info

Protein Target Info (Source: Uniprot.org)

Uniprot Id P0DMV8/P0DMV9
Gene Name Hspa1b
Protein Name heat shock protein family A (Hsp70) member 1A/1B
Tissue Specificity HSPA1B is testis-specific.
Alternative Names Heat shock 70 kDa protein 1A; Heat shock 70 kDa protein 1B
Subcellular Localization Cytoplasm.

*if product is indicated to react with multiple species, protein info is based on the human gene.

Ontology

Protein 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 (PubMed:24012426, PubMed:26865365, PubMed:24318877). 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 (PubMed:27708256). 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 (PubMed:27137183). Enhances STUB1-mediated SMAD3 ubiquitination and degradation and facilitates STUB1-mediated inhibition of TGF-beta signaling (PubMed:24613385). Essential for STUB1-mediated ubiquitination and degradation of FOXP3 in regulatory T-cells (Treg) during inflammation (PubMed:23973223). Negatively regulates heat shock- induced HSF1 transcriptional activity during the attenuation and recovery phase period of the heat shock response (PubMed:9499401).
Research Areas Alfalfa, Flax, Human, Maize, Plant

*You can search these to find other products in these research areas.
Background HSP70 genes encode abundant heat-inducible 70-kDa HSPs (HSP70s). In most eukaryotes HSP70 genes exist as part of a multigene family. They are found in most cellular compartments of eukaryotes including nuclei, mitochondria, chloroplasts, the endoplasmic reticulum and the cytosol, as well as in bacteria. The genes show a high degree of conservation, having at least 5O% identity. The N-terminal two thirds of HSP70s are more conserved than the C-terminal third. HSP70 binds ATP with high affinity and possesses a weak ATPase activity which can be stimulated by binding to unfolded proteins and synthetic peptides. When HSC70 (constitutively expressed) present in mammalian cells was truncated, ATP binding activity was found to reside in an N-terminal fragment of 44kDa which lacked peptide binding capacity. Polypeptide binding ability therefore resided within the C-terminal half. The structure of this ATP binding domain displays multiple features of nucleotide binding proteins. All HSP70s, regardless of location, bind proteins, particularly unfolded ones. The molecular chaperones of the HSP70 family recognize and bind to nascent polypeptide chains as well as partially folded intermediates of proteins preventing their aggregation and misfolding. The binding of ATP triggers a critical conformational change leading to the release of the bound substrate protein. The universal ability of HSP70s to undergo cycles of binding to and release from hydrophobic stretches of partially unfolded proteins determines their role in a great variety of vital intracellular functions such as protein synthesis, protein folding and oligomerization and protein transport.

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EK7115
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Customer Q&As

  • Q: I found bubbles in the wells of my ELISA kit. Is this normal or will it have an impact on the performance of the kit?
    A: The bubbles in the wells is an uncommon issue, but it would not affect the results of your experiment.
    We suggest to wash the plate to remove the bubbles as suggested in the protocol below prior to the assay:
    1. Wash the plate 3 times with the 1X wash buffer.
    2. Discard the liquid in the wells into an appropriate waste receptacle. Then, invert the plate on the benchtop onto a paper towel and tap the plate to gently blot any remaining liquid. It is recommended that the wells are not allowed to completely dry at any time.
    3. Add 300 µl of the 1x wash buffer to each assay well. (For cleaner background incubate for 60 seconds between each wash).
    4. Repeat steps a-b 2 additional times.
  • Q: There are precipitates in the sample diluent buffer. What is it?
    A: The precipitates are fatty substances contained in BSA. It would not affect the performance of the kit. If you want to confirm this, please contact Boster Support.
  • Q: I left this product out in ambient temperatures for around 5 days, will the kit be stable?
    A: Our kits should still be stable after being in ambient temperatures for around 5 days. It would not affect the performance of the kit. We suggest you to run the test and contact us if does not work as expected.
  • Q: What is the number of density of cells recommended for ELISA assay (for cell culture supernants and cell lysates)?
    A: The number of cells we recommened for cell culture supernants and cell lysates is 10^6 cells/mL.
  • Q: How many samples can I test on one ELISA plate?
    A: On each 96 well plate, we recommend running an 8-point standard curve with duplicate wells. With the remaining 80 wells, 40 samples can be tested in duplicate.
  • Q: The volumes of my samples are small, would it be possible to use 50µl/sample?
    A: There are two solutions for insufficient sample volume. <br> 1. Dilute the samples with the provided sample diluent buffer into 100ul. <br> 2. If 50ul of sample is added into a well, add 50ul of standard solution.
  • Q: Will this kit work on tissue samples?
    A: For tissue homogenates, endogenous biotin should be considered. Endogenous biotin in tissue homogenates might introduce false positive signal. Please run the test as described below to confirm if there is Endogenous biotin in the tissue lysate samples using the ELISA kit. <br> • Add tissue homogenates into the wells and then add ABC and TMB without adding any biotinylated detection antibody to see if any signal will be observed. <br> • If no signal is produced, then you can work on the tissue sample by using the kit.
  • Q: Is it possible to dilute 10X TBS stock that is 250mM Tris Base/Tris-Hcl, 27 mM KCl, and 1.37M NaCl to be used as the wash buffer?
    A: You can dilute the 10X TBS stock to 1XTBS and test the pH value of the 1xTBS. <br> The 1XTBS can be used if the pH value falls in the range of 7.2-7.6.
  • Q: Would your kits work normally with samples from Wistar Rat?
    A: Our kits would work normally with samples from Wistar Rat.
  • Q: How can I store the dissolved wash buffer for a longer period of time?
    A: You can prepare a concentrated wash buffer (25X) which can be stored at 4°C for 1-3 months.
  • Q: Does the TMB color developing agent contain H2O2?
    A: Yes, the TMB color developing agent contain H2O2.
  • Q: Does this ELISA kit contain any product produced in humans or primates? <br> Keywords: component, ingredient
    A: None of the components in our ELISA kits are produced in humans or primates.
  • Q: What is the volume of the recombinant protein control? What is its shelf life? <br>Keywords: expiration, storage, temperature, size
    A: The volume of the control is around 100pg. If unopened, the shelf life of the control is the same as the whole kit - "Store at 4˚C for 6 months, at -20˚C for 12 months." The reconstituted control can be stored at -20˚C for 2 days.
  • Q: Will it be okay to run the experiment if I accidentally used the wrong buffer (e.g. sample diluent buffer) for antibody dilution instead of the antibody diluent buffer? Keywords: dilution, replace, substitute, sample buffer
    A: Using the sample diluent buffer for antibody dilution instead of the antibody diluent buffer will negatively affect the test result to some extent. The values of both standard and sample might be lower than the normal values.
  • Q: The protocol says to use neutral PBS and provides a recipe. Could I use neutral PBS made with KCl or KH2PO4 (common constituents for most PBS recipes) instead? Keywords: protocol, alternative buffer, applications, reagent recipe
    A: Yes, it is ok to use common PBS recipes. We have tried many types of buffers with common constituents, and no significant difference was observed whatsoever.
  • Q: Why are my O.D. values different than your values on the datasheet? Keyword: troubleshooting, reference
    A: We detected the kit in our lab and got our values on the datasheet before delivery, but protein activity will decrease as time goes on, so you may get lower O.D. values than ours. However, it should still be in a reasonable range and the standards can be used to calculate sample values. Good linearity of curve is more important than the actual numerical value.
  • Q: On the ELISA kit datasheet it says "HRP substrate TMB was used to visualize HRP enzymatic reaction." What is HRP and which part of the kit contains HRP? Keyword: kit components, reagents, protocol
    A: It means Avidin-Biotin-Peroxidase Complex(AR1103) and you could find it in Kit Components.
  • Q: Do you offer any ELISA kits that can work with whole blood samples instead of plasma or urine? Keyword: applications
    A: We test serum and plasma routinely, and there is very little difference between serum, plasma and whole blood. The whole blood also contains proteins we need to test. The kit can be used to test whole blood in theory.
  • Q: Does silicic acid (formula SiOH4) affect the results of the ELISA assay? Keyword: protocol, reagents
    A: The acid may affect the binding of antigen to antibody, it is not recommended to use. That being said, it is unlikely to affect the reaction if the solution remains an overall neutral environment.
  • Q: Is there lot-to-lot variation of the ELISA kits? What are Boster's general services when I have questions about your kits? Keyword: technical support, help, customer service
    A: The NIBSC/WHO 1st International Standard is evaluated, we always test our kits before delivery, and customers can find the test result on the protocol. If the customer needs technical support from us to analyze their data, please contact us and we ask that you provide us the following information: the lot# and production date, and when did the customer detect the kit.
  • Q: The results of my standards do not look correct, what could be the problem? Keyword: troubleshoot, protocol
    A: Double check the protocol for plate washing. It should be examined on three aspects: 1) Did you make the washing buffer correctly? 2) How long did you let the buffer stay in wells? 3) What was the volume of the washing buffer added to each well? In addition, pay attention when adding sample to avoid contamination. And we suggest testing the standards again. Values of standards at low concentration are more affected than that at higher concentration, so customer can still get expected values at high concentration even if there is an error. If problems persist, please contact technical support with the catalog and lot number of your product.
  • Q: What is the well depth of the 96 well plates for the ELISA kits? Keyword: well capacity, product size
    A: The well depth is 300ul, and the max capacity is 350ul. The height of the well is 1.1 cm, and the internal size is 1 cm.
  • Q: The kit does not include wash solution, what should I do? Keyword: wash buffer
    A: Our Elisa kits do not come with wash solution, but we have included information about how to make washing buffer on the datasheet. Please refer to the "Material Required But Not Provided" section. We also offer washing buffer for sale separately (Phosphate Buffered Saline Powder SKU: AR0030).
  • Q: For your ELISA kits, are the capture and detection antibodies polyclonals or monoclonals? Keyword: antibody clonality
    A: This information can be found for each kit under the "Properties" section, and you can find the immunogen sequence information in the "Overview" section.
  • Q: Do your ELISA kits come with sealants or plate covers? Keyword: storage, sequential use
    A: The kit can be used within a month sequentially if it's opened and stored at 4 degree. There is no need to use sealants, the plate can be packaged with aluminum foil bag, and for other reagents keep bottle tightly closed.
  • Q: What is the concentration of Sodium Azide in your Elisa kits? Keyword: preservative
    A: Our Elisa kits contain 0.02% Sodium Azide. All of the components except TMB colour developing reagent and stop solution contains 0.02% Sodium azide as the preservative.
  • Q: Are there positive and negative controls available for my ELISA kit? Keyword: positive control, negative control
    A: We can provide a recombinant protein as a control. It costs $50 per control and takes 2 weeks to manufacture. We cannot provide a positive or negative control in serum.
  • Q: Why do I get positive results for my knockout (KO) model when used as a control?
    A: The knockout (KO) model may contain truncated forms of the target protein which can be detected by the ELISA assay.
  • Q: How long do I soak my plate in the wash buffer?
    A: The plate should soak in 0.3mL PBS or TBS wash buffer for at least 1-2 minutes in an automated wash station.
  • Q: Can I use Tween in my wash buffer?
    A: While it is not recommended to use Tween in your wash buffer, small amounts (<0.1% concentration) may decrease background due to insufficient blocking.
  • Q: What plate type do I use to set up the microplate reader?
    A: Our plates are made with the Corning costar plates similar to the DNA-BIND 96 -well plates.
  • Q: What should I use for negative control?
    A: Please contact us for negative control suggestions. You can also check expression databases such as genecards, uniprot etc. Due to logistic reasons, we do not sell serum or lysates that we use internally for positive or negative control.
  • Q: Where can I find troubleshooting information? What should I do if I have unexpected bands, high background, no signal, weak signal
    A: You can find Boster's troubleshoot guides under tech support tab. Please contact us for further assistance on troubleshooting your experiment.
  • Q: What is the normal level of this protein in my sample of interest?
    A: We have reviewed literature and have gathered this information for most of our ELISA kits. You can find this information on the product page or contact us if you cannot find it. However this information is only suggestive and cannot replace pilot studies in determining the optimal sample dilution ratio.
  • Q: Is the plate separable? Can I use only part of the kit and save the rest for later? How many samples can I run with one kit?
    A: Yes the plate is separable if the kit says that there are removable strips. There are 12 strips of 8 wells each, all removable from the plate. The amount of samples you can run depend on a few factors. In the most common ELISA set up, you will use two strips for standards, and 10 strips for samples using duplicates, which let you run up to 40 samples per kit. Contact us if you have questions regarding other situations.
  • Q: Does the kit contain sample preparation reagents? How do I prepare my samples?
    A: Since different sample types require different reagents, we do not include them in the ELISA kit. However we do have each reagent mentioned in the file below available at very reasonable prices. Be sure to check them out. For sample preparation protocols please download the file below: https://www.bosterbio.com/media/pdf/Boster_Sample_Preparation_Protocols.pdf
  • Q: Can this ELISA kit work on brain tissue homogenate, cell culture supernatant, saliva, urine, serum, whole blood or any other sample type?
    A: In theory the ELISA kit will work for all sample types if the target protein is present at a level that falls within the linear range of the ELISA kit detection range. We guarantee the kit to work on the sample types that we have tested. If you want dilution ratio suggestions on these sample types please contact our technical support. For sample types that we have not tested for, we suggest you run pilot experiments to decide the optimal sample dilution.
  • Q: Can this ELISA kit react with the pro-form of the target protein? Can this ELISA kit react with an isoform of the protein?
    A: In general, unless otherwise specified, the ELISA kit is pan-specific, meaning that it will react with all different forms of the target protein if they share the majority of the target protein's sequence. The capture and detection antibodies are reactive to the entire sequence of the standard protein. You can find the sequence information of the standard protein in the "immunogen" section. For more information about the specificity of the kit for your particular experiment, please contact our technical support.
  • Q: Can this ELISA kit react with human, mouse, rat or other species?
    A: If the kit is reactive to another commonly used species (human, mouse, and/or rat), we would have listed it as a separate product. If you want to check cross-reactivity to a species that is not included in the 3 species listed above, please contact our technical support for more information. As a rule of thumb, if the sequences are 90%+ identical, there is a high chance of cross-reactivity for your species of interest.
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