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Rat Interferon-induced, double-stranded RNA-activated protein kinase (Eif2ak2) ELISA Kit

SKU:
RTEB0689
Product Type:
ELISA Kit
Size:
96 Assays
Uniprot:
Q63184
Range:
0.312-20 ng/mL
ELISA Type:
Sandwich
Synonyms:
Eif2ak2, PRKR, PRKRA
Reactivity:
Rat
€649
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Description

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Product Name:Rat Interferon-induced, double-stranded RNA-activated protein kinase (Eif2ak2) ELISA Kit
Product Code:RTEB0689
Alias:Interferon-induced, double-stranded RNA-activated protein kinase, Eukaryotic translation initiation factor 2-alpha kinase 2, eIF-2A protein kinase 2, Interferon-inducible RNA-dependent protein kinase, Protein kinase RNA-activated, PKR, Tyrosine-protein kinase EIF2AK2, Eif2ak2, Prkr, 2.7.11.1, 2.7.10.2
Uniprot:Q63184
Reactivity:Rat
Range:0.312-20 ng/mL
Detection Method:Sandwich
Size:96 Assay
Storage:Please see kit components below for exact storage details
Note:For research use only
UniProt Protein Function: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 west nile virus (WNV), sindbis virus (SV), foot-and-mouth virus (FMDV), semliki Forest virus (SFV) and lymphocytic choriomeningitis virus (LCMV). 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 and IRS1. 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. Regulates proliferation, differentiation and survival of hematopoietic stem/progenitor cells and induction of cytokines and chemokines
UniProt Protein Details:

By similarity. Plays a role in cortex-dependent memory consolidation. Ref.3

Catalytic activity: ATP + a protein = ADP + a phosphoprotein.ATP + a [protein]-L-tyrosine = ADP + a [protein]-L-tyrosine phosphate.

Enzyme regulation: Initially produced in an inactive form and is activated by binding to viral dsRNA, which causes dimerization and autophosphorylation in the activation loop and stimulation of function. ISGylation can activate it in the absence of viral infection. Can also be activated by heparin, proinflammatory stimuli, growth factors, cytokines, oxidative stress and the cellular protein PRKRA. Activity is markedly stimulated by manganese ions. Activation is blocked by the cellular proteins TARBP2, DUS2L, NPM1, NCK1 and ADAR

By similarity.

Subunit structure: Homodimer

By similarity. Interacts with DNAJC3

By similarity. Interacts with STRBP. Forms a complex with FANCA, FANCC, FANCG and HSP70

By similarity. Interacts with ADAR/ADAR1

By similarity. The inactive form interacts with NCK1 and GSN. Interacts (via the kinase catalytic domain) with STAT3 (via SH2 domain), TRAF2 (C-terminus), TRAF5 (C-terminus) and TRAF6 (C-terminus). Interacts with MAP2K6, IKBKB/IKKB, IRS1, NPM1, TARBP2, NLRP1, NLRP3, NLRC4 and AIM2. Interacts (via DRBM 1 domain) with DUS2L (via DRBM domain). Interacts with DHX9 (via N-terminus) and this interaction is dependent upon activation of the kinase

By similarity. Ref.2

Subcellular location: Cytoplasm

By similarity. Nucleus

By similarity. Cytoplasm › perinuclear region

By similarity.

Induction: By type I interferons

By similarity.

Post-translational modification: Autophosphorylated on several Ser, Thr and Tyr residues. Autophosphorylation of Thr-411 is dependent on Thr-406 and is stimulated by dsRNA binding and dimerization. Autophosphorylation apparently leads to the activation of the kinase. Tyrosine autophosphorylation is essential for efficient dsRNA-binding, dimerization, and kinase activation

By similarity.

Sequence similarities: Belongs to the protein kinase superfamily. Ser/Thr protein kinase family. GCN2 subfamily.Contains 2 DRBM (double-stranded RNA-binding) domains.Contains 1 protein kinase domain.

NCBI Summary:double-stranded RNA dependent eukaryotic initiation factor 2-kinase [RGD, Feb 2006]
UniProt Code:Q63184
NCBI GenInfo Identifier:9506993
NCBI Gene ID:54287
NCBI Accession:NP_062208.1
UniProt Related Accession:Q63184
Molecular Weight:29.9kDa
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  
NCBI Protein Information:interferon-induced, double-stranded RNA-activated protein kinase; eIF-2A protein kinase 2; protein kinase RNA-activated; tyrosine-protein kinase EIF2AK2; interferon-inducible RNA-dependent protein kinase; 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; Protein kinase RNA-activated; PKR; Tyrosine-protein kinase EIF2AK2
UniProt Gene Name:Eif2ak2  
UniProt Entry Name:E2AK2_RAT
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.

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