Human Cell Biology ELISA Kits 1
Human HSP-70 (Heat Shock Protein 70) ELISA Kit (HUES02815)
- SKU:
- HUES02815
- Product Type:
- ELISA Kit
- Size:
- 96 Assays
- Uniprot:
- P38646
- Sensitivity:
- 0.47ng/mL
- Range:
- 0.78-50ng/mL
- ELISA Type:
- Sandwich
- Synonyms:
- HSPA9, CSA, GRP-75, GRP75, HSPA9B, MOT, MOT2, MTHSP75, PBP74
- Reactivity:
- Human
- Sample Type:
- Serum, plasma and other biological fluids
- Research Area:
- Cell Biology
Description
| Assay type: | Sandwich |
| Format: | 96T |
| Assay time: | 4.5h |
| Reactivity: | Human |
| Detection Method: | Colormetric |
| Detection Range: | 0.78-50 ng/mL |
| Sensitivity: | 0.47 ng/mL |
| Sample Volume Required Per Well: | 100µL |
| Sample Type: | Serum, plasma and other biological fluids |
| Specificity: | This kit recognizes Human HSP-70 in samples. No significant cross-reactivity or interference between Human HSP-70 and analogues was observed. |
This ELISA kit uses Sandwich-ELISA as the method. The micro ELISA plate provided in this kit has been pre-coated with an antibody specific to Human HSP-70. Standards or samples are added to the appropriate micro ELISA plate wells and combined with the specific antibody. Then a biotinylated detection antibody specific for Human HSP-70 and Avidin-Horseradish Peroxidase (HRP) conjugate are added to each micro plate well successively and incubated. Free components are washed away. The substrate solution is added to each well. Only those wells that contain Human HSP-70, biotinylated detection antibody and Avidin-HRP conjugate will appear blue in color. The enzyme-substrate reaction is terminated by adding Stop Solution and the color turns yellow. The optical density (OD) is measured spectrophotometrically at a wavelength of 450 nm ± 2 nm. The OD value is proportional to the concentration of Human HSP-70. The concentration of Human HSP-70 in samples can be calculated by comparing the OD of the samples to the standard curve.
| UniProt Protein Function: | HSPA9B: Implicated in the control of cell proliferation and cellular aging. May also act as a chaperone. Interacts with FXN. Interacts with HSCB. Component of the MINOS/MitOS complex, that includes IMMT, HSPA9 and CHCHD3 and associates with mitochondrial outer membrane proteins SAMM50, MTX1 and MTX2. Belongs to the heat shock protein 70 family. |
| UniProt Protein Details: | Protein type:Nucleolus; Heat shock protein; Chaperone Chromosomal Location of Human Ortholog: 5q31. 1 Cellular Component: focal adhesion; mitochondrion; cytoplasm; nucleolus Molecular Function:protein binding; ubiquitin protein ligase binding; unfolded protein binding; ATP binding Biological Process: cellular protein metabolic process; protein folding; protein export from nucleus; protein targeting to mitochondrion; negative regulation of apoptosis |
| NCBI Summary: | This gene encodes a member of the heat shock protein 70 gene family. The encoded protein is primarily localized to the mitochondria but is also found in the endoplasmic reticulum, plasma membrane and cytoplasmic vesicles. This protein is a heat-shock cognate protein. This protein plays a role in cell proliferation, stress response and maintenance of the mitochondria. A pseudogene of this gene is found on chromosome 2. [provided by RefSeq, May 2010] |
| UniProt Code: | P38646 |
| NCBI GenInfo Identifier: | 21264428 |
| NCBI Gene ID: | 3313 |
| NCBI Accession: | P38646. 2 |
| UniProt Secondary Accession: | P38646,P30036, P31932, Q1HB43, Q53H23, Q6GU03, Q9BWB7 Q9UC56, B2RCM1, |
| UniProt Related Accession: | P38646 |
| Molecular Weight: | 679 |
| NCBI Full Name: | Stress-70 protein, mitochondrial |
| NCBI Synonym Full Names: | heat shock 70kDa protein 9 (mortalin) |
| NCBI Official Symbol: | HSPA9 |
| NCBI Official Synonym Symbols: | CSA; MOT; MOT2; CRP40; GRP75; PBP74; GRP-75; HSPA9B; MTHSP75; HEL-S-124m |
| NCBI Protein Information: | stress-70 protein, mitochondrial; mortalin-2; p66-mortalin; mortalin, perinuclear; heat shock 70kD protein 9B; peptide-binding protein 74; 75 kDa glucose-regulated protein; catecholamine-regulated protein 40; epididymis secretory sperm binding protein Li 124m |
| UniProt Protein Name: | Stress-70 protein, mitochondrial |
| UniProt Synonym Protein Names: | 75 kDa glucose-regulated protein; GRP-75; Heat shock 70 kDa protein 9; Mortalin; MOT; Peptide-binding protein 74; PBP74 |
| Protein Family: | Stress-70 protein |
| UniProt Gene Name: | HSPA9 |
| UniProt Entry Name: | GRP75_HUMAN |
As the OD values of the standard curve may vary according to the conditions of the actual assay performance (e. g. operator, pipetting technique, washing technique or temperature effects), the operator should establish a standard curve for each test. Typical standard curve and data is provided below for reference only.
| Concentration (ng/mL) | O.D | Average | Corrected |
| 50 | 2.384 2.39 | 2.387 | 2.299 |
| 25 | 1.596 1.636 | 1.616 | 1.528 |
| 12.5 | 0.96 0.952 | 0.956 | 0.868 |
| 6.25 | 0.467 0.483 | 0.475 | 0.387 |
| 3.13 | 0.276 0.25 | 0.263 | 0.175 |
| 1.56 | 0.202 0.174 | 0.188 | 0.1 |
| 0.78 | 0.138 0.142 | 0.14 | 0.052 |
| 0 | 0.087 0.089 | 0.088 | -- |
Precision
Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Human HSP-70 were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human HSP-70 were tested on 3 different plates, 20 replicates in each plate.
| Intra-assay Precision | Inter-assay Precision | |||||
| Sample | 1 | 2 | 3 | 1 | 2 | 3 |
| n | 20 | 20 | 20 | 20 | 20 | 20 |
| Mean (ng/mL) | 2.30 | 6.00 | 22.60 | 2.30 | 5.60 | 24.10 |
| Standard deviation | 0.10 | 0.30 | 1.10 | 0.10 | 0.30 | 0.90 |
| C V (%) | 4.35 | 5.00 | 4.87 | 4.35 | 5.36 | 3.73 |
Recovery
The recovery of Human HSP-70 spiked at three different levels in samples throughout the range of the assay was evaluated in various matrices.
| Sample Type | Range (%) | Average Recovery (%) |
| Serum (n=5) | 93-108 | 100 |
| EDTA plasma (n=5) | 91-106 | 97 |
| Cell culture media (n=5) | 91-103 | 98 |
Linearity
Samples were spiked with high concentrations of Human HSP-70 and diluted with Reference Standard & Sample Diluent to produce samples with values within the range of the assay.
| Serum (n=5) | EDTA plasma (n=5) | Cell culture media (n=5) | ||
| 1:2 | Range (%) | 84-96 | 89-101 | 89-106 |
| Average (%) | 91 | 94 | 96 | |
| 1:4 | Range (%) | 103-119 | 80-92 | 90-104 |
| Average (%) | 108 | 86 | 98 | |
| 1:8 | Range (%) | 93-107 | 85-95 | 92-108 |
| Average (%) | 100 | 90 | 99 | |
| 1:16 | Range (%) | 100-115 | 83-93 | 93-106 |
| Average (%) | 108 | 87 | 101 |
An unopened kit can be stored at 4°C for 1 month. If the kit is not used within 1 month, store the items separately according to the following conditions once the kit is received.
| Item | Specifications | Storage |
| Micro ELISA Plate(Dismountable) | 8 wells ×12 strips | -20°C, 6 months |
| Reference Standard | 2 vials | |
| Concentrated Biotinylated Detection Ab (100×) | 1 vial, 120 µL | |
| Concentrated HRP Conjugate (100×) | 1 vial, 120 µL | -20°C(shading light), 6 months |
| Reference Standard & Sample Diluent | 1 vial, 20 mL | 4°C, 6 months |
| Biotinylated Detection Ab Diluent | 1 vial, 14 mL | |
| HRP Conjugate Diluent | 1 vial, 14 mL | |
| Concentrated Wash Buffer (25×) | 1 vial, 30 mL | |
| Substrate Reagent | 1 vial, 10 mL | 4°C(shading light) |
| Stop Solution | 1 vial, 10 mL | 4°C |
| Plate Sealer | 5 pieces | |
| Product Description | 1 copy | |
| Certificate of Analysis | 1 copy |
- Set standard, test sample and control (zero) wells on the pre-coated plate and record theirpositions. It is recommended to measure each standard and sample in duplicate. Note: addall solutions to the bottom of the plate wells while avoiding contact with the well walls. Ensuresolutions do not foam when adding to the wells.
- Aliquot 100µl of standard solutions into the standard wells.
- Add 100µl of Sample / Standard dilution buffer into the control (zero) well.
- Add 100µl of properly diluted sample (serum, plasma, tissue homogenates and otherbiological fluids) into test sample wells.
- Cover the plate with the sealer provided in the kit and incubate for 90 min at 37°C.
- Aspirate the liquid from each well, do not wash. Immediately add 100µL of BiotinylatedDetection Ab working solution to each well. Cover the plate with a plate seal and gently mix. Incubate for 1 hour at 37°C.
- Aspirate or decant the solution from the plate and add 350µL of wash buffer to each welland incubate for 1-2 minutes at room temperature. Aspirate the solution from each well andclap the plate on absorbent filter paper to dry. Repeat this process 3 times. Note: a microplatewasher can be used in this step and other wash steps.
- Add 100µL of HRP Conjugate working solution to each well. Cover with a plate seal andincubate for 30 min at 37°C.
- Aspirate or decant the solution from each well. Repeat the wash process for five times asconducted in step 7.
- Add 90µL of Substrate Reagent to each well. Cover with a new plate seal and incubate forapproximately 15 min at 37°C. Protect the plate from light. Note: the reaction time can beshortened or extended according to the actual color change, but not by more than 30min.
- Add 50 µL of Stop Solution to each well. Note: Adding the stop solution should be done inthe same order as the substrate solution.
- Determine the optical density (OD value) of each well immediately with a microplate readerset at 450 nm.
Related Products
Rat HSP-70/HSPA9 (Heat Shock Protein 70) ELISA Kit
