SKU:EK7111
Pack Size:96wells/kit, with removable strips.
Validated Species:Human
Application:ELISA
Sensitivity:1.73 ng/ml
Assay Range:11-700 ng/ml
Sample Type:Cell lysates, Serum, Tissue
Technical
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Components
Gene
Information
Application
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Customer
Q&As

Introduction

Boster's ELISA Kit is for the detection of Hsp60 in cell lysates, tissue extracts, and serum samples. Each kit contains sufficient components to quantitate the Hsp60 concentration in up to 40 samples, tested in duplicate.

Overview

Product Name HSP60 ELISA Kit
SKU/Catalog Number EK7111
Description Colorimetric detection of HSP60. 96wells/kit, with removable strips.
Cite This Product HSP60 ELISA Kit (Boster Biological Technology, Pleasanton CA, USA, Catalog # EK7111)
Host
Validated Species 194
Predicted Species

*This antibody is predicted to react with the above species based on antigen sequence similarities. Our Boster Guarantee covers the use of this product with the above species.

Application 18

*Our Boster Guarantee covers the use of this product in the above tested applications.

**For positive and negative control design, consult "Tissue specificity" under Protein Target Info.

Recommended Detection Systems
*Blocking peptide can be purchased at $50. Contact us for more information
**Boster also offers various secondary antibodies for Immunoflourescecne and IHC. Take advantage of the buy 1 primary antibody get 1 secondary antibody for free promotion for the entire year 2018!
Immunogen
Conjugate
Cross Reactivity There is no detectable cross-reactivity.
Pack Size 96wells/kit, with removable strips.

Properties

Clonality
Clone Number
Sensitivity 1.73 ng/ml
*Sensitivity, or Lower Limit of Detection (LLD), is the minimum level of target protein the ELISA assay can detect. We measure 20 blank wells and if the O.D. value is 2 standard deviations higher than the blanks' average O.D. the sample can be deemed positive.
Assay Range 11-700 ng/ml
*This assay range is determined using common samples. For samples with low target protein concentrations, users can adjust the standard curve to extend the lower limit of assay range.
Sample Type Cell lysates, Serum, Tissue

*The above listed samples are the ones valided with the assay. If you do not see your sample of interest listed, as long as there is enough level of target protein present in the sample, this Picokine™ ELISA kit should detect it.
**For protocol and tips regarding preparing your sample of interest, please check our ELISA sample preparation guide.
Capture Antibody
Detection Antibody
Normal Levels Some research articles suggesting the natural levels of HSPD1 are listed below:
Form
Contents
Concentration
Activating Reagent
Storage Store at 4°C.
Purification
Isotype

Kit Components

DescriptionQuantity
Anti-Hsp60 Immunoassay Plate1 Plate
5X Hsp60 Extraction Reagent1 vial/10 ml
Recombinant Hsp60 Standard1 vials/700ng
Standard and Sample Diluent1 vial/ 50 ml
10X Wash Buffer Concentrate1 vial/100 ml
Anti-Hsp60 Biotinylated Antibody Concentrate1 vial/150 μl
Anti-Hsp60 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

Material Required But Not Provided

1. Ultra pure water.

2. Additional reagents and materials for cell lysate and tissue extract preparation, including protease inhibitors.

3. Precision pipettors, with disposable plastic tips.

4. Polypropylene or polyethylene tubes to prepare samples − do not use polystyrene, polycarbonate or glass tubes.

5. A container to prepare 1X Wash Buffer.

6. A wash bottle or an automated 96-well plate washer.

7. Disposable reagent reservoirs.

8. A standard microtiter plate reader for measuring absorbance at 450 nm.

9. Adhesive plate sealers.

Protein Target Info (Source: Uniprot.org)

You can check the tissue specificity below for information on selecting positive and negative control.

Gene Name HSPD1
Protein Name 60 kDa heat shock protein, mitochondrial
Molecular Weight
Protein Function Chaperonin implicated in mitochondrial protein import and macromolecular assembly. Together with Hsp10, facilitates the correct folding of imported proteins. May also prevent misfolding and promote the refolding and proper assembly of unfolded polypeptides generated under stress conditions in the mitochondrial matrix (PubMed:1346131, PubMed:11422376). The functional units of these chaperonins consist of heptameric rings of the large subunit Hsp60, which function as a back-to-back double ring. In a cyclic reaction, Hsp60 ring complexes bind one unfolded substrate protein per ring, followed by the binding of ATP and association with 2 heptameric rings of the co-chaperonin Hsp10. This leads to sequestration of the substrate protein in the inner cavity of Hsp60 where, for a certain period of time, it can fold undisturbed by other cell components. Synchronous hydrolysis of ATP in all Hsp60 subunits results in the dissociation of the chaperonin rings and the release of ADP and the folded substrate protein (Probable).
Tissue Specificity
Sequence Similarities
Subcellular Localization Mitochondrion matrix.
Uniprot ID P10809
Alternative Names 60 kDa heat shock protein, mitochondrial; 60 kDa chaperonin; Chaperonin 60; CPN60; Heat shock protein 60; HSP-60; Hsp60; HuCHA60; Mitochondrial matrix protein P1; P60 lymphocyte protein; HSPD1; HSP60
Reseach Areas 2446,2481,2577,2452,2423,2320,2366,2367|
*if product is indicated to react with multiple species, protein info is based on the human gene.

Background for 60 kDa heat shock protein, mitochondrial

In both prokaryotic and eukaryotic cells, the misfolding and aggregation of proteins during biogenesis and under conditions of cellular stress are prevented by molecular chaperones. Members of the HSP60 family of heat shock proteins are some of the best characterized chaperones. HSP60, also known as Cpn60 or GroEl, is an abundant protein synthesized constitutively in the cell that is induced to a higher concentration after brief cell shock. It is present in many species and exhibits a remarkable sequence homology among various counterparts in bacteria, plants, and mammals with more than half of the residues identical between bacterial and mammalian HSP60. Whereas mammalian HSP60 is localized within the mitochondria, plant HSP60, or otherwise known as Rubisco-binding protein, is located in plant chloroplasts. It has been indicated that these proteins carry out a very important biological function due to the fact that HSP60 is present in so many different species. The common characteristics of the HSP60s from the divergent species are i) high abundance, ii) induction with environmental stress such as heat shock, iii) homo-oligomeric structures of either 7 or 14 subunits which reversibly dissociate in the presence of Mg2+ and ATP,iv) ATPase activity and v) a role in folding and assembly of oligomeric protein structures. These similarities are supported by recent studies where the single-ring human mitochondrial homolog, HSP60 with its co-chaperonin, HSP10 were expressed in a E. coli strain, engineered so that the groE operon is under strict regulatory control. This study has demonstrated that expression of HSP60-HSP10 was able to carry out all essential in vivo functions of GroEL and its co-chaperonin, GroES. Another important function of HSP60 and HSP10 is their protective functions against infection and cellular stress. HSP60 has however been linked to a number of autoimmune diseases, as well as Alzheimers, coronary artery diseases, MS, and diabetes.

Intra/Inter Assay Precision

Sample Dilution

1. Samples must first be diluted prior to testing.

2. Prepare at least 250 µL of sample in Standard and Sample Diluent. Mix samples well prior to analysis.

3. Suggested starting dilutions for samples:
o For cell and tissue lysates, prepare a 200 µg/mL total protein concentration dilution in Standard and Sample Diluent.
o When measuring Hsp60 in serum samples, use a 1:1 dilution in 50mM Acetic Acid (pH 4.67) and assay total 100 µL sample volume directly i.e. add 50 µl of 50 mM Acetic Acid to all standard and sample wells followed by 50 µL of appropriately diluted standards or samples to each well. Run each standard, sample, or blank in duplicate. Follow with the remaining procedure. Expect lower OD values, however, serum matrix effects are removed.

Note: If values for samples are not within the range of the standard curve, optimal sample dilutions need to be determined.

Standard Preparation

1. Reconstitute standard vial with 1 mL of Standard and Sample Diluent for a concentration of 700 ng/mL. Mix well.

2. Label seven (7) tubes, one for each additional standard curve point: 350 ng/mL, 175 ng/mL, 88 ng/mL, 44 ng/mL, 22 ng/mL, 11 ng/ml, and 0 ng/mL.

3. Pipet 250 µl of Standard and Sample Diluent into each tube.

4. Serial dilute the 700 ng/mL standard 1:1 with Standard and Sample Diluent. Perform dilution by mixing 250 µL of the previous standard with 250 µL of Standard and Sample Diluent. Continue until reach the standard value of 11 ng/ mL.

5. Use Standard and Sample Diluent only as the zero standard value.

1X Wash Buffer Preparation

Prepare 1X wash buffer by adding 100 ml of Wash Buffer Concentrate to 900 ml deionized or distilled water to prepare 1000 mL of Wash Buffer.

Biotinylated Antibody Working Solution Preparation

It is recommended to prepare this reagent immediately prior to use by diluting the Avidin-BiotinPeroxidase Complex 1:100 with Avidin-Biotin-Peroxidase Diluent. Prepare 100 µl by adding 1 µl of Avidin-Biotin-Peroxidase Complex to 99 µl of Avidin-Biotin-Peroxidase Diluent for each well. Mix gently and thoroughly and use within 2 hours of generation.

Streptavidin-HRP Working Solution Preparation

It is recommended to prepare this reagent immediately prior to use by diluting Streptavidin-HRP Concentrate 1:100 with Streptavidin-HRP Diluent. Prepare 100 µl by adding 1 µl of Avidin-Biotin-Peroxidase Complex to 99 µl of Avidin-Biotin-Peroxidase Diluent for each well. Mix gently and thoroughly and use within 2 hours of generation.

Assay Overview

1. Prepare Standard and samples in Standard and Sample Diluent.

2. Add 100 µL of Standard or sample to appropriate wells.

3. Cover plate with Plate Sealer and incubate at 20°C-25°C for 1 hour.

4. Wash plate four times with 1X Wash Buffer.

5. Add 100 µL of Biotinylated Antibody Working Solution to each well.

6. Cover plate with Plate Sealer and incubate at 20°C-25°C for 1 hour.

7. Wash plate four times with 1X Wash Buffer.

8. Add 100 µL of Streptavidin-HRP Working Solution to each well.

9. Cover plate with Plate Sealer and incubate at room temperature for 30 minutes.

10. Wash plate four times with 1X Wash Buffer.

11. Add 100 µL of TMB Substrate to each well..

12. Develop the plate in the dark at room temperature for 30 minutes.

13. Stop reaction by adding 100 µL of Stop Solution to each well.

14. Measure absorbance on a plate reader at 450 nm.

Colorimetric detection of HSP60. 96wells/kit, with removable strips.
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EK7111
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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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