HSP70 ELISA Kit

Catalog No. ABIN2964827

This Mouse, Rat, Human, Dog and Monkey HSP70 ELISA Kit is a Colorimetric ELISA Kit designed to quantify Mouse, Rat, Human, Dog and Monkey HSP70. This ELISA Kit has been cited in 4+ publications.

Quick Overview for HSP70 ELISA Kit (ABIN2964827)

Target: HSP70 (Heat Shock Protein 70 (HSP70))

Reactivity: Mouse, Rat, Human, Dog, Monkey

Detection Method: Colorimetric

Method Type: Sandwich ELISA

Detection Range: 0.42 ng/mL - 27 ng/mL

Application: ELISA

Sample Type: Serum, Plasma, Cell Lysate, Tissue Samples

Product Details HSP70 ELISA Kit

Minimum Detection Limit: 0.42 ng/mL

Purpose: High-Sensitivity Colorimetric detection of HSP70

Analytical Method: Quantitative

Sensitivity: 0.038 ng/mL

Characteristics: ELISA kit used to quantitate HSP70 concentration in samples.

Components:

• Anti-Hsp70 Immunoassay Plate
• Recombinant Hsp70 Standard
• Standard and Sample Diluent
• 10X Wash Buffer Concentrate
• Anti-Hsp70 Biotinylated Antibody Concentrate
• Anti-Hsp70 Biotinylated Antibody Diluent
• Streptavidin: HRP Concentrate
• Streptavidin: HRP Diluent
• TMB Substrate
• Stop Solution

Material not included: - Ultra pure water
- Additional reagents and materials for cell lysate and tissue extract preparation, including protease inhibitors
- Precision pipettors, with disposable plastic tips
- Polypropylene or polyethylene tubes to prepare samples − do not use polystyrene, polycarbonate or glass tubes
- A container to prepare 1X Wash Buffer
- A wash bottle or an automated 96-well plate washer

Application Details

Assay Time: 0.5 h

Plate: Pre-coated

Protocol:

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 37 °C for 2 hours.
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 at 37 °C for 2 hours.
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 37 °C 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.

Sample Preparation:

Sample Handling
All blood components and biological materials should be handled as potentially hazardous. Follow universal precautions when handling and disposing of infectious agents.
100 µl of diluted sample is required per well.
Samples must be assayed in duplicate each time the assay is performed.
Samples should be frozen if not analyzed shortly after harvest. For long-term storage, aliquot and freeze samples. Avoid repeated freeze-thaw cycles when storing samples.
If particulate is present in samples, centrifuge prior to analysis.
If the integrity of the sample is of concern, make a note on the Plate Template and interpret results with caution.

Sample Dilution
Samples must first be diluted prior to testing.
Suggested starting dilutions for samples:
- For native human cell lysates, dilute samples 1:128 in Standard and Sample
Diluent. For example, dilute 2 µL of sample in 254 µL Standard and Sample
Diluent. Mix well. For non-human cell lysates, dilute samples 1:4 in Standard and Sample Diluent.
- For serum and plasma samples, dilute samples 1:4 in Standard and Sample Diluent. For example, dilute 60 µL of sample in 180 µL Standard and Sample Diluent. Mix well.
Note: If values for samples are not within the range of the standard curve, optimal sample dilutions need to be determined.

Prepare at least 240 µL of sample in Standard and Sample Diluent. Mix samples well prior to analysis.
Note: The cell lysate Extraction Reagent is not included with this kit. This kit is mainly for use with serum and plasma samples.

Assay Procedure:

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 37 °C for 2 hours.
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 at 37 °C for 2 hours.
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 37 °C 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.

Calculation of Results:

Duplicate absorbance values should be within 10% of each other. Care should be taken when interpreting data with differences in absorbance values greater than 10%.

1. Prepare a standard curve to determine the amount of Hsp70 in an unknown sample. Plot the average absorbance obtained for each standard concentration on the vertical (Y) axis versus the corresponding Hsp70 concentration on the horizontal (X) axis using graph paper or curve-fitting software.

2. Calculate the Hsp70 concentration in unknown samples using the prepared standard curve. Determine the amount of Hsp70 in each unknown sample by noting the Hsp70 concentration (X axis) that correlates with the absorbance value (Y axis) obtained for the unknown sample.

3. Multiply the Hsp70 concentration obtained by the dilution factor to determine the amount of Hsp70 in the undiluted sample.

Restrictions: For Research Use only

Handling

Storage: 4 °C

References for HSP70 ELISA Kit (ABIN2964827)

Kavanagh, Davis, Jenkins, Flynn: "Effects of heated hydrotherapy on muscle HSP70 and glucose metabolism in old and young vervet monkeys." in: Cell stress & chaperones, Vol. 21, Issue 4, pp. 717-25, (2016) (PubMed).

Kanouchi, Kakimoto, Nakano, Suzuki, Nakai, Shiozaki, Akikoka, Otomaru, Nagano, Matsumoto: "The Brewed Rice Vinegar Kurozu Increases HSPA1A Expression and Ameliorates Cognitive Dysfunction in Aged P8 Mice." in: PLoS ONE, Vol. 11, Issue 3, pp. e0150796, (2016) (PubMed).

Miyazaki, Nakamura, Hineno, Kobayashi, Kinoshita, Yoshida, Ikeda: "Elevation of serum heat-shock protein levels in amyotrophic lateral sclerosis." in: Neurological sciences : official journal of the Italian Neurological Society and of the Italian Society of Clinical Neurophysiology, Vol. 37, Issue 8, pp. 1277-81, (2016) (PubMed).

Chichester, Wylie, Craft, Kavanagh: "Muscle heat shock protein 70 predicts insulin resistance with aging." in: The journals of gerontology. Series A, Biological sciences and medical sciences, Vol. 70, Issue 2, pp. 155-62, (2015) (PubMed).

Target Details for HSP70

Target: HSP70 (Heat Shock Protein 70 (HSP70))

Alternative Name: HSP70

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 44 kDa 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.