Description
Human Interferon-induced, double-stranded RNA-activated protein kinase (EIF2AK2) ELISA Kit
The Human Interferon-Induced Double-Stranded RNA-Activated Protein Kinase (EIF2AK2) ELISA Kit is specifically designed for the precise measurement of EIF2AK2 levels in human samples including serum, plasma, and cell culture supernatants. This kit offers exceptional sensitivity and specificity, ensuring accurate and consistent results for a variety of research applications.EIF2AK2, also known as Protein Kinase R (PKR), is a key player in the innate immune response to viral infections. Activation of EIF2AK2 leads to the phosphorylation of eIF2α, resulting in the inhibition of protein synthesis to hinder viral replication.
Dysregulation of EIF2AK2 has been implicated in various diseases, including viral infections, autoimmune disorders, and cancer, highlighting its importance as a biomarker for disease progression and therapeutic development.Overall, the Human Interferon-Induced Double-Stranded RNA-Activated Protein Kinase (EIF2AK2) ELISA Kit offers researchers a reliable tool for studying the role of EIF2AK2 in immune responses and disease pathogenesis, ultimately contributing to the advancement of therapeutic strategies.
| Product Name: | Human Interferon-induced, double-stranded RNA-activated protein kinase (EIF2AK2) ELISA Kit |
| SKU: | HUEB1152 |
| Size: | 96T |
| Target: | Human Interferon-induced, double-stranded RNA-activated protein kinase (EIF2AK2) |
| Synonyms: | Eukaryotic translation initiation factor 2-alpha kinase 2, Interferon-inducible RNA-dependent protein kinase, P1/eIF-2A protein kinase, Protein kinase RNA-activated, Tyrosine-protein kinase EIF2AK2, p68 kinase, eIF-2A protein kinase 2, PKR, PKR, PRKR |
| Assay Type: | Sandwich |
| Detection Method: | ELISA |
| Reactivity: | Human |
| Detection Range: | 0.156-10ng/mL |
| Sensitivity: | 0.045ng/mL |
| Intra CV: | 5.0% | ||||||||||||||||||||
| Inter CV: | 9.8% | ||||||||||||||||||||
| Linearity: |
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| Recovery: |
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| Function: | IFN-induced dsRNA-dependent serine/threonine-protein kinase which plays a key role in the innate immune response to viral infection and is also involved in the regulation of signal transduction, apoptosis, cell proliferation and differentiation. Exerts its antiviral activity on a wide range of DNA and RNA viruses including hepatitis C virus (HCV), hepatitis B virus (HBV), measles virus (MV) and herpes simplex virus 1 (HHV-1). Inhibits viral replication via phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (EIF2S1), this phosphorylation impairs the recycling of EIF2S1 between successive rounds of initiation leading to inhibition of translation which eventually results in shutdown of cellular and viral protein synthesis. Also phosphorylates other substrates including p53/TP53, PPP2R5A, DHX9, ILF3, IRS1 and the HHV-1 viral protein US11. In addition to serine/threonine-protein kinase activity, also has tyrosine-protein kinase activity and phosphorylates CDK1 at 'Tyr-4' upon DNA damage, facilitating its ubiquitination and proteosomal degradation. Either as an adapter protein and/or via its kinase activity, can regulate various signaling pathways (p38 MAP kinase, NF-kappa-B and insulin signaling pathways) and transcription factors (JUN, STAT1, STAT3, IRF1, ATF3) involved in the expression of genes encoding proinflammatory cytokines and IFNs. Activates the NF-kappa-B pathway via interaction with IKBKB and TRAF family of proteins and activates the p38 MAP kinase pathway via interaction with MAP2K6. Can act as both a positive and negative regulator of the insulin signaling pathway (ISP). Negatively regulates ISP by inducing the inhibitory phosphorylation of insulin receptor substrate 1 (IRS1) at 'Ser-312' and positively regulates ISP via phosphorylation of PPP2R5A which activates FOXO1, which in turn up-regulates the expression of insulin receptor substrate 2 (IRS2). Can regulate NLRP3 inflammasome assembly and the activation of NLRP3, NLRP1, AIM2 and NLRC4 inflammasomes. Can trigger apoptosis via FADD-mediated activation of CASP8. Plays a role in the regulation of the cytoskeleton by binding to gelsolin (GSN), sequestering the protein in an inactive conformation away from actin. |
| Uniprot: | P19525 |
| Sample Type: | Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids |
| Specificity: | Natural and recombinant human Interferon-induced, double-stranded RNA-activated protein kinase |
| Sub Unit: | (Microbial infection) Interacts with human herpes simplex virus 1 (HHV-1) protein US11 in an RNA-dependent manner (PubMed:11836380). The inactive form interacts with Toscana virus (TOS) NSS (PubMed:23325696). Interacts with herpes virus 8 protein v-IRF2; this interaction inhibits EIF2AK2 activation (PubMed:11160738). Interacts with vaccinia protein E3 (PubMed:25740987). |
| Research Area: | Cancer |
| Subcellular Location: | Cytoplasm Nucleus Cytoplasm Perinuclear region Nuclear localization is elevated in acute leukemia, myelodysplastic syndrome (MDS), melanoma, breast, colon, prostate and lung cancer patient samples or cell lines as well as neurocytes from advanced Creutzfeldt-Jakob disease patients. |
| Storage: | Please see kit components below for exact storage details |
| Note: | For research use only |
| 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 |
| Component | Quantity (96 Assays) | Storage |
| ELISA Microplate (Dismountable) | 8×12 strips | -20°C |
| Lyophilized Standard | 2 | -20°C |
| Sample Diluent | 20ml | -20°C |
| Assay Diluent A | 10mL | -20°C |
| Assay Diluent B | 10mL | -20°C |
| Detection Reagent A | 120µL | -20°C |
| Detection Reagent B | 120µL | -20°C |
| Wash Buffer | 30mL | 4°C |
| Substrate | 10mL | 4°C |
| Stop Solution | 10mL | 4°C |
| Plate Sealer | 5 | - |
Other materials and equipment required:
- Microplate reader with 450 nm wavelength filter
- Multichannel Pipette, Pipette, microcentrifuge tubes and disposable pipette tips
- Incubator
- Deionized or distilled water
- Absorbent paper
- Buffer resevoir
*Note: The below protocol is a sample protocol. Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.
Allow all reagents to reach room temperature (Please do not dissolve the reagents at 37°C directly). All the reagents should be mixed thoroughly by gently swirling before pipetting. Avoid foaming. Keep appropriate numbers of strips for 1 experiment and remove extra strips from microtiter plate. Removed strips should be resealed and stored at -20°C until the kits expiry date. Prepare all reagents, working standards and samples as directed in the previous sections. Please predict the concentration before assaying. If values for these are not within the range of the standard curve, users must determine the optimal sample dilutions for their experiments. We recommend running all samples in duplicate.
| Step | |
| 1. | Add Sample: Add 100µL of Standard, Blank, or Sample per well. The blank well is added with Sample diluent. Solutions are added to the bottom of micro ELISA plate well, avoid inside wall touching and foaming as possible. Mix it gently. Cover the plate with sealer we provided. Incubate for 120 minutes at 37°C. |
| 2. | Remove the liquid from each well, don't wash. Add 100µL of Detection Reagent A working solution to each well. Cover with the Plate sealer. Gently tap the plate to ensure thorough mixing. Incubate for 1 hour at 37°C. Note: if Detection Reagent A appears cloudy warm to room temperature until solution is uniform. |
| 3. | Aspirate each well and wash, repeating the process three times. Wash by filling each well with Wash Buffer (approximately 400µL) (a squirt bottle, multi-channel pipette,manifold dispenser or automated washer are needed). Complete removal of liquid at each step is essential. After the last wash, completely remove remaining Wash Buffer by aspirating or decanting. Invert the plate and pat it against thick clean absorbent paper. |
| 4. | Add 100µL of Detection Reagent B working solution to each well. Cover with the Plate sealer. Incubate for 60 minutes at 37°C. |
| 5. | Repeat the wash process for five times as conducted in step 3. |
| 6. | Add 90µL of Substrate Solution to each well. Cover with a new Plate sealer and incubate for 10-20 minutes at 37°C. Protect the plate from light. The reaction time can be shortened or extended according to the actual color change, but this should not exceed more than 30 minutes. When apparent gradient appears in standard wells, user should terminatethe reaction. |
| 7. | Add 50µL of Stop Solution to each well. If color change does not appear uniform, gently tap the plate to ensure thorough mixing. |
| 8. | Determine the optical density (OD value) of each well at once, using a micro-plate reader set to 450 nm. User should open the micro-plate reader in advance, preheat the instrument, and set the testing parameters. |
| 9. | After experiment, store all reagents according to the specified storage temperature respectively until their expiry. |
When carrying out an ELISA assay it is important to prepare your samples in order to achieve the best possible results. Below we have a list of procedures for the preparation of samples for different sample types.
| Sample Type | Protocol |
| Serum | If using serum separator tubes, allow samples to clot for 30 minutes at room temperature. Centrifuge for 10 minutes at 1,000x g. Collect the serum fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. If serum separator tubes are not being used, allow samples to clot overnight at 2-8°C. Centrifuge for 10 minutes at 1,000x g. Remove serum and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. |
| Plasma | Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples at 4°C for 15 mins at 1000 × g within 30 mins of collection. Collect the plasma fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. Note: Over haemolysed samples are not suitable for use with this kit. |
| Urine & Cerebrospinal Fluid | Collect the urine (mid-stream) in a sterile container, centrifuge for 20 mins at 2000-3000 rpm. Remove supernatant and assay immediately. If any precipitation is detected, repeat the centrifugation step. A similar protocol can be used for cerebrospinal fluid. |
| Cell culture supernatant | Collect the cell culture media by pipette, followed by centrifugation at 4°C for 20 mins at 1500 rpm. Collect the clear supernatant and assay immediately. |
| Cell lysates | Solubilize cells in lysis buffer and allow to sit on ice for 30 minutes. Centrifuge tubes at 14,000 x g for 5 minutes to remove insoluble material. Aliquot the supernatant into a new tube and discard the remaining whole cell extract. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C. |
| Tissue homogenates | The preparation of tissue homogenates will vary depending upon tissue type. Rinse tissue with 1X PBS to remove excess blood & homogenize in 20ml of 1X PBS (including protease inhibitors) and store overnight at ≤ -20°C. Two freeze-thaw cycles are required to break the cell membranes. To further disrupt the cell membranes you can sonicate the samples. Centrifuge homogenates for 5 mins at 5000xg. Remove the supernatant and assay immediately or aliquot and store at -20°C or -80°C. |
| Tissue lysates | Rinse tissue with PBS, cut into 1-2 mm pieces, and homogenize with a tissue homogenizer in PBS. Add an equal volume of RIPA buffer containing protease inhibitors and lyse tissues at room temperature for 30 minutes with gentle agitation. Centrifuge to remove debris. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C. |
| Breast Milk | Collect milk samples and centrifuge at 10,000 x g for 60 min at 4°C. Aliquot the supernatant and assay. For long term use, store samples at -80°C. Minimize freeze/thaw cycles. |
