Human Developmental Biology ELISA Kits
Human SOCS3 (Suppressors of Cytokine Signaling 3) ELISA Kit (HUES02999)
- SKU:
- HUES02999
- Product Type:
- ELISA Kit
- Size:
- 96 Assays
- Uniprot:
- O14543
- Sensitivity:
- 9.38pg/mL
- Range:
- 15.63-1000pg/mL
- ELISA Type:
- Sandwich
- Synonyms:
- ATOD4, CIS3, Cish3, MGC71791, SOCS-3, SSI-3, SSI3,
- Reactivity:
- Human
- Sample Type:
- Serum, plasma and other biological fluids
- Research Area:
- Developmental Biology
Description
| Assay type: | Sandwich |
| Format: | 96T |
| Assay time: | 4.5h |
| Reactivity: | Human |
| Detection Method: | Colormetric |
| Detection Range: | 15.63-1000 pg/mL |
| Sensitivity: | 9.38 pg/mL |
| Sample Volume Required Per Well: | 100µL |
| Sample Type: | Serum, plasma and other biological fluids |
| Specificity: | This kit recognizes Human SOCS3 in samples. No significant cross-reactivity or interference between Human SOCS3 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 SOCS3. 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 SOCS3 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 SOCS3, 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 SOCS3. The concentration of Human SOCS3 in samples can be calculated by comparing the OD of the samples to the standard curve.
| UniProt Protein Function: | SOCS3: SOCS family proteins form part of a classical negative feedback system that regulates cytokine signal transduction. SOCS3 is involved in negative regulation of cytokines that signal through the JAK/STAT pathway. Inhibits cytokine signal transduction by binding to tyrosine kinase receptors including gp130, LIF, erythropoietin, insulin, IL12, GCSF and leptin receptors. Binding to JAK2 inhibits its kinase activity. Suppresses fetal liver erythropoiesis. Regulates onset and maintenance of allergic responses mediated by T-helper type 2 cells. Regulates IL-6 signaling in vivo. Probable substrate recognition component of a SCF-like ECS (Elongin BC- CUL2/5-SOCS-box protein) E3 ubiquitin-protein ligase complex which mediates the ubiquitination and subsequent proteasomal degradation of target proteins. Seems to recognize IL6ST. Interacts with multiple activated proteins of the tyrosine kinase signaling pathway including IGF1 receptor, insulin receptor and JAK2. Binding to JAK2 is mediated through the KIR and SH2 domains to a phosphorylated tyrosine residue within the JAK2 JH1 domain. Binds specific activated tyrosine residues of the leptin, EPO, IL12, GSCF and gp130 receptors. Interaction with CSNK1E stabilizes SOCS3 protein. Component of the probable ECS(SOCS3) E3 ubiquitin-protein ligase complex which contains CUL5, RNF7/RBX2, Elongin BC complex and SOCS3. Interacts with CUL5, RNF7, TCEB1 and TCEB2. Interacts with CUL2. Interacts with FGFR3. Interacts with INSR. Widely expressed with high expression in heart, placenta, skeletal muscle, peripheral blood leukocytes, fetal and adult lung, and fetal liver and kidney. Lower levels in thymus. |
| UniProt Protein Details: | Protein type:Inhibitor Chromosomal Location of Human Ortholog: 17q25. 3 Cellular Component: cytoplasm; cytosol Molecular Function:protein binding; protein kinase inhibitor activity Biological Process: regulation of growth; negative regulation of inflammatory response; cytokine and chemokine mediated signaling pathway; protein ubiquitination; negative regulation of protein kinase activity; negative regulation of insulin receptor signaling pathway; JAK-STAT cascade; positive regulation of cell differentiation; negative regulation of JAK-STAT cascade; negative regulation of apoptosis Disease: Dermatitis, Atopic, 4 |
| NCBI Summary: | This gene encodes a member of the STAT-induced STAT inhibitor (SSI), also known as suppressor of cytokine signaling (SOCS), family. SSI family members are cytokine-inducible negative regulators of cytokine signaling. The expression of this gene is induced by various cytokines, including IL6, IL10, and interferon (IFN)-gamma. The protein encoded by this gene can bind to JAK2 kinase, and inhibit the activity of JAK2 kinase. Studies of the mouse counterpart of this gene suggested the roles of this gene in the negative regulation of fetal liver hematopoiesis, and placental development. [provided by RefSeq, Jul 2008] |
| UniProt Code: | O14543 |
| NCBI GenInfo Identifier: | 20178094 |
| NCBI Gene ID: | 9021 |
| NCBI Accession: | O14543. 1 |
| UniProt Secondary Accession: | O14543,O14509, |
| UniProt Related Accession: | O14543 |
| Molecular Weight: | |
| NCBI Full Name: | Suppressor of cytokine signaling 3 |
| NCBI Synonym Full Names: | suppressor of cytokine signaling 3 |
| NCBI Official Symbol: | SOCS3 |
| NCBI Official Synonym Symbols: | CIS3; SSI3; ATOD4; Cish3; SSI-3; SOCS-3 |
| NCBI Protein Information: | suppressor of cytokine signaling 3; STAT-induced STAT inhibitor 3; cytokine-inducible SH2 protein 3 |
| UniProt Protein Name: | Suppressor of cytokine signaling 3 |
| UniProt Synonym Protein Names: | Cytokine-inducible SH2 protein 3; CIS-3; STAT-induced STAT inhibitor 3; SSI-3 |
| Protein Family: | Suppressor of cytokine signaling |
| UniProt Gene Name: | SOCS3 |
| UniProt Entry Name: | SOCS3_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 (pg/mL) | O.D | Average | Corrected |
| 1000 | 2.415 2.419 | 2.417 | 2.364 |
| 500 | 1.675 1.701 | 1.688 | 1.635 |
| 250 | 0.948 0.942 | 0.945 | 0.892 |
| 125 | 0.421 0.445 | 0.433 | 0.38 |
| 62.5 | 0.254 0.248 | 0.251 | 0.198 |
| 31.25 | 0.169 0.141 | 0.155 | 0.102 |
| 15.63 | 0.097 0.115 | 0.106 | 0.053 |
| 0 | 0.045 0.061 | 0.053 | -- |
Precision
Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Human SOCS3 were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human SOCS3 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 (pg/mL) | 50.17 | 115.98 | 487.30 | 47.09 | 105.87 | 524.86 |
| Standard deviation | 2.52 | 6.32 | 19.78 | 2.88 | 5.91 | 17.90 |
| C V (%) | 5.02 | 5.45 | 4.06 | 6.12 | 5.58 | 3.41 |
Recovery
The recovery of Human SOCS3 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) | 94-106 | 100 |
| EDTA plasma (n=5) | 91-106 | 99 |
| Cell culture media (n=5) | 87-97 | 92 |
Linearity
Samples were spiked with high concentrations of Human SOCS3 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 (%) | 97-113 | 84-96 | 90-104 |
| Average (%) | 104 | 90 | 95 | |
| 1:4 | Range (%) | 88-99 | 83-94 | 85-98 |
| Average (%) | 93 | 89 | 90 | |
| 1:8 | Range (%) | 93-107 | 83-95 | 83-97 |
| Average (%) | 98 | 89 | 89 | |
| 1:16 | Range (%) | 88-102 | 82-93 | 82-96 |
| Average (%) | 93 | 86 | 87 |
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.
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