Description
Human EIF2a (Eukaryotic Translation Initiation Factor 2 Alpha) ELISA Kit
The Human EIF2A (Eukaryotic Translation Initiation Factor 2 Alpha) ELISA Kit is a powerful tool for the precise measurement of EIF2A levels in human samples such as serum, plasma, and cell culture supernatants. This kit provides exceptional sensitivity and specificity to ensure accurate and consistent results, making it suitable for a wide range of research applications.EIF2A is a key component of the protein synthesis machinery, playing a crucial role in the initiation of translation and regulation of gene expression. Dysregulation of EIF2A has been implicated in various diseases, including cancer, neurodegenerative disorders, and metabolic conditions, highlighting its importance as a potential therapeutic target and diagnostic marker.
By using the Human EIF2A ELISA Kit, researchers can gain valuable insights into the role of EIF2A in disease pathology and potential treatment strategies. Its reliability and precision make it an indispensable tool for studying molecular mechanisms and developing novel interventions in the field of translational research.
| Assay type: | Sandwich |
| Format: | 96T |
| Assay time: | 4.5h |
| Reactivity: | Human |
| Detection Method: | Colormetric |
| Detection Range: | 78.13-5000 pg/mL |
| Sensitivity: | 46.88 pg/mL |
| Sample Volume Required Per Well: | 100µL |
| Sample Type: | Serum, plasma and other biological fluids |
| Specificity: | This kit recognizes Human EIF2a in samples. No significant cross-reactivity or interference between Human EIF2a 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 EIF2a. 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 EIF2a 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 EIF2a, 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 EIF2a. The concentration of Human EIF2a in samples can be calculated by comparing the OD of the samples to the standard curve.
| UniProt Protein Function: | PKR: a protein kinase of the PEK family. Upon binding double-stranded RNA, it becomes autophosphorylated and activated. Phosphorylates and inhibits the alpha subunit of eIF2 alpha, which leads to an inhibition of the initiation of protein synthesis. Controls the activation of several transcription factors such as NF-kappaB, p53 and Stats. Mediates apoptosis induced by many different stimuli, such as LPS, TNF-alpha, viral infection and serum starvation. |
| UniProt Protein Details: | Protein type:Translation; Kinase, protein; EC 2. 7. 10. 2; Protein kinase, Other; Protein kinase, Ser/Thr (non-receptor); EC 2. 7. 11. 1; Other group; PEK family Chromosomal Location of Human Ortholog: 2p22-p21 Cellular Component: membrane; perinuclear region of cytoplasm; cytoplasm; ribosome; nucleus; cytosol Molecular Function:protein serine/threonine kinase activity; protein binding; double-stranded RNA binding; non-membrane spanning protein tyrosine kinase activity; protein phosphatase type 2A regulator activity; ATP binding; eukaryotic translation initiation factor 2alpha kinase activity; protein kinase activity Biological Process: positive regulation of cytokine production; peptidyl-tyrosine phosphorylation; translation; activation of MAPKK activity; transcription, DNA-dependent; unfolded protein response; response to virus; protein amino acid autophosphorylation; viral infectious cycle; protein amino acid phosphorylation; positive regulation of stress-activated MAPK cascade; positive regulation of chemokine production; evasion by virus of host immune response; activation of NF-kappaB transcription factor; negative regulation of cell proliferation; modification by virus of host cellular process; negative regulation of viral genome replication; virus-host interaction; negative regulation of translation; innate immune response; negative regulation of osteoblast proliferation; defense response to virus; negative regulation of apoptosis |
| NCBI Summary: | The protein encoded by this gene is a serine/threonine protein kinase that is activated by autophosphorylation after binding to dsRNA. The activated form of the encoded protein can phosphorylate translation initiation factor EIF2S1, which in turn inhibits protein synthesis. This protein is also activated by manganese ions and heparin. Three transcript variants encoding two different isoforms have been found for this gene. [provided by RefSeq, Oct 2011] |
| UniProt Code: | P19525 |
| NCBI GenInfo Identifier: | 125527 |
| NCBI Gene ID: | 5610 |
| NCBI Accession: | P19525. 2 |
| UniProt Secondary Accession: | P19525,Q52M43, Q7Z6F6, Q9UIR4, A8K3P0, D6W584, E9PC80 |
| UniProt Related Accession: | P19525 |
| Molecular Weight: | 62,094 Da |
| NCBI Full Name: | Interferon-induced, double-stranded RNA-activated protein kinase |
| NCBI Synonym Full Names: | eukaryotic translation initiation factor 2-alpha kinase 2 |
| NCBI Official Symbol: | EIF2AK2 |
| NCBI Official Synonym Symbols: | PKR; PRKR; EIF2AK1; PPP1R83 |
| NCBI Protein Information: | interferon-induced, double-stranded RNA-activated protein kinase; p68 kinase; eIF-2A protein kinase 2; P1/eIF-2A protein kinase; tyrosine-protein kinase EIF2AK2; interferon-inducible elF2alpha kinase; double stranded RNA activated protein kinase; protein phosphatase 1, regulatory subunit 83; protein kinase, interferon-inducible double stranded RNA dependent |
| UniProt Protein Name: | Interferon-induced, double-stranded RNA-activated protein kinase |
| UniProt Synonym Protein Names: | Eukaryotic translation initiation factor 2-alpha kinase 2; eIF-2A protein kinase 2; Interferon-inducible RNA-dependent protein kinase; P1/eIF-2A protein kinase; Protein kinase RNA-activated; PKR; Tyrosine-protein kinase EIF2AK2 (EC:2. 7. 10. 2); p68 kinase |
| UniProt Gene Name: | EIF2AK2 |
| UniProt Entry Name: | E2AK2_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 |
| 5000 | 2.464 2.468 | 2.466 | 2.377 |
| 2500 | 1.673 1.731 | 1.702 | 1.613 |
| 1250 | 1.002 0.97 | 0.986 | 0.897 |
| 625 | 0.491 0.517 | 0.504 | 0.415 |
| 312.5 | 0.322 0.296 | 0.309 | 0.22 |
| 156.25 | 0.21 0.186 | 0.198 | 0.109 |
| 78.13 | 0.144 0.144 | 0.144 | 0.055 |
| 0 | 0.086 0.092 | 0.089 | -- |
Precision
Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Human EIF2a were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human EIF2a 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) | 235.80 | 462.53 | 2372.67 | 236.88 | 467.40 | 2244.43 |
| Standard deviation | 12.21 | 20.91 | 125.99 | 15.73 | 25.66 | 120.75 |
| C V (%) | 5.18 | 4.52 | 5.31 | 6.64 | 5.49 | 5.38 |
Recovery
The recovery of Human EIF2a 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) | 92-105 | 98 |
| EDTA plasma (n=5) | 86-99 | 91 |
| Cell culture media (n=5) | 88-102 | 96 |
Linearity
Samples were spiked with high concentrations of Human EIF2a 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 (%) | 85-99 | 92-107 | 91-105 |
| Average (%) | 92 | 99 | 98 | |
| 1:4 | Range (%) | 91-105 | 79-91 | 85-97 |
| Average (%) | 97 | 85 | 91 | |
| 1:8 | Range (%) | 86-98 | 81-94 | 87-102 |
| Average (%) | 92 | 88 | 94 | |
| 1:16 | Range (%) | 87-101 | 86-101 | 89-102 |
| Average (%) | 93 | 92 | 94 |
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.
