Rat Cell Biology ELISA Kits 2

Rat Eukaryotic translation initiation factor 2-alpha kinase 3 (Eif2ak3) ELISA Kit

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SKU:
RTEB0696
Product Type:
ELISA Kit
Size:
96 Assays
Uniprot:
Q9Z1Z1
ELISA Type:
Sandwich
Synonyms:
PERK, PRKR-like endoplasmic reticulum kinase, Pancreatic eIF2-alpha kinase
Reactivity:
Rat

Description

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Product Name:Rat Eukaryotic translation initiation factor 2-alpha kinase 3 (Eif2ak3) ELISA Kit
Product Code:RTEB0696
Alias:Eukaryotic translation initiation factor 2-alpha kinase 3, PRKR-like endoplasmic reticulum kinase, Pancreatic eIF2-alpha kinase, Eif2ak3, Pek, Perk, 2.7.11.1
Uniprot:Q9Z1Z1
Reactivity:Rat
Range:Please contact us for more information
Detection Method:Sandwich
Size:96 Assay
Storage:Please see kit components below for exact storage details
Note:For research use only
UniProt Protein Function:PERK: a transmembrane protein kinase of the PEK family resident in the endoplasmic reticulum (ER) membrane and is linked to insulin processing. Couples ER stress to translation inhibition. Stress induces autophosphorylation of its kinase domain and increases its activity. Phosphorylates the alpha subunit of eukaryotic translation-initiation factor 2 (eIF2alpha), leading to its inactivation and thus to a rapid reduction of translational initiation and repression of global protein synthesis. A critical effector of unfolded protein response (UPR)-induced G1 growth arrest due to the loss of cyclin d1. Forms dimers with BiP in resting cells. Oligomerizes in ER-stressed cells. LOF mutations cause Wolcott-Rallison syndrome (WRS), characterized by insulin-dependent diabetes in early infancy and, later, multiple system abnormalities. Neuronal death in Alzheimer?s and Parkinson?s diseases is thought to be due to ER stress and has been weakly linked to PEK.
UniProt Protein Details:

Protein type:Translation; Kinase, protein; Protein kinase, Ser/Thr (non-receptor); EC 2.7.11.1; Protein kinase, Other; Membrane protein, integral; Other group; PEK family; PEK subfamily

Cellular Component: cytoplasm; endoplasmic reticulum; endoplasmic reticulum membrane; integral to membrane; membrane; perinuclear region of cytoplasm

Molecular Function:ATP binding; enzyme binding; eukaryotic translation initiation factor 2alpha kinase activity; Hsp90 protein binding; identical protein binding; protein kinase activity; protein phosphatase binding; protein serine/threonine kinase activity

Biological Process: angiogenesis; bone mineralization; calcium-mediated signaling; caspase activation; cellular response to amino acid starvation; cellular response to glucose starvation; chondrocyte development; endocrine pancreas development; endoplasmic reticulum organization and biogenesis; ER overload response; fat cell differentiation; insulin-like growth factor receptor signaling pathway; lactation; negative regulation of apoptosis; negative regulation of myelination; negative regulation of translation; negative regulation of translation in response to stress; ossification; pancreas development; peptidyl-serine phosphorylation; positive regulation of protein binding; positive regulation of signal transduction; positive regulation of transcription from RNA polymerase I promoter; protein amino acid autophosphorylation; protein amino acid phosphorylation; protein homooligomerization; regulation of fatty acid metabolic process; skeletal development; SREBP-mediated signaling pathway; translation; unfolded protein response

NCBI Summary:kinase that phosphorylates eukaryotic initiation factor 2alpha; has an important role in translational control [RGD, Feb 2006]
UniProt Code:Q9Z1Z1
NCBI GenInfo Identifier:13928816
NCBI Gene ID:29702
NCBI Accession:NP_113787.1
UniProt Related Accession:Q9Z1Z1
Molecular Weight:124,770 Da
NCBI Full Name:eukaryotic translation initiation factor 2-alpha kinase 3
NCBI Synonym Full Names:eukaryotic translation initiation factor 2 alpha kinase 3
NCBI Official Symbol:Eif2ak3  
NCBI Official Synonym Symbols:PEK  
NCBI Protein Information:eukaryotic translation initiation factor 2-alpha kinase 3
UniProt Protein Name:Eukaryotic translation initiation factor 2-alpha kinase 3
UniProt Synonym Protein Names:PRKR-like endoplasmic reticulum kinase; Pancreatic eIF2-alpha kinase
Protein Family:Eukaryotic translation initiation factor 2-alpha kinase
UniProt Gene Name:Eif2ak3  
UniProt Entry Name:E2AK3_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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