Catalog # Aliquot Size

E1111G05 5 g E1111G10 10 g

EIF2AK3, Active Recombinant human protein expressed in E.coli cells

Catalog # E1111G

Lot # G336 -2

Product Description Specific Activity

Recombinant human EIF2AK3 (563-1115) was expressed in E. coli cells using an N-terminal GST tag. The 120,000 accession number is NM_004836. 90,000 Gene Aliases 60,000

PERK, PEK, WRS, HRI, DKFZp781H1925 30,000 (cpm) Activity Formulation 0 0 200 400 600 800 Recombinant protein stored in 50mM Tris-HCl, pH 7.5, Protein (ng) 150mM NaCl, 10mM glutathione, 0.1mM EDTA, 0.25mM DTT, 0.1mM PMSF, 25% glycerol. The specific activity of EIF2AK3 was determined to be 19 nmol /min/mg as per activity assay protocol.

Storage and Stability Purity

o Store product at –70 C. For optimal storage, aliquot target into smaller quantities after centrifugation and store at recommended temperature. For most favorable performance, avoid repeated handling and multiple freeze/thaw cycles. The purity of EIF2AK3 was determined to be ~90% by densitometry, approx. MW 115kDa . Scientific Background

EIF2AK3 phosphorylates the alpha subunit of eukaryotic translation-initiation factor 2 (EIF2) leading to its inactivation and a rapid reduction of translational initiation and repression of global protein synthesis. EIF2AK3 is a type I membrane protein located in the EIF2AK3, Active endoplasmic reticulum (ER), where it is induced by ER Recombinant human protein expressed in E. coli cells stress caused by malfolded proteins (1). EIF2AK3 plays a major role in the ability of cells to adapt to ER stress and is Catalog # E11-11G also involved in an integrated adaptive response to Specific Activity 19 nmol/min/mg hypoxic stress in HeLa cells (2). EIF2AK3 functions in iron Lot # G336-2 homeostasis and may play a role in hemolytic and Purity ~90% inflammatory anemia. Concentration 0.1 µ g/ µl Stability 1yr at –70 oC from date of shipment Storage & Shipping Store product at –70 oC. For optimal storage, References aliquot target into smaller quantities after centrifugation and store at recommended 1. Harding, H. P. et.al: EIF2AK3 is essential for translational temperature. For most favorable performance, regulation and cell survival during the unfolded protein avoid repeated handling and multiple response. Molec. Cell 5: 897-904, 2000. freeze/thaw cycles. Product shipped on dry ice. 2. Blais, J. D. et.al: Activating transcription factor 4 is translationally regulated by hypoxic stress. Molec. Cell. Biol. 24: 7469-7482, 2004.

To place your order, please contact us by phone 1-(604)-232-4600, fax 1-604-232-4601 or by email: [email protected] www.signalchem.com

FOR IN VITRO RESEARCH PURPOSES ONLY. NOT INTENDED FOR USE IN HUMAN OR ANIMALS. Activity Assay Protocol

Reaction Components

Active (Catalog #: E11-11G) [33 P]-ATP Assay Cocktail Active EIF2AK3 (0.1 µg/ µl) diluted with Kinase Dilution Prepare 250 µM [33 P]-ATP Assay Cocktail in a designated Buffer III (Catalog #: K23-09 ) and assayed as outlined in radioactive working area by adding the following sample activity plot. (Note: these are suggested working components: 150 µl of 10mM ATP Stock Solution (Catalog #: dilutions and it is recommended that the researcher A50-09 ), 100 µl [ 33 P]-ATP (1mCi/100 µl), 5.75ml of Kinase Assay perform a serial dilution of Active EIF2AK3 for optimal Buffer I (Catalog #: K01-09 ). Store 1ml aliquots at –20 oC. results). Kinase Dilution Buffer III (Catalog #: K23 -09 ) 10mM ATP Stock Solution (Catalog #: A50-09 ) Kinase Assay Buffer I (Catalog #: K01-09 ) diluted at a Prepare ATP stock solution by dissolving 55mg of ATP in 10ml 1:4 ratio (5X dilution) with final 50ng/ µl BSA solution. of Kinase Assay Buffer I (Catalog #: K01-09 ). Store 200 µl aliquots at –20 oC.

Kinase Assay Buffe r I (Catalog #: K01 -09 ) Substrate (Catalog #: S12-30G ) Buffer components: 25mM MOPS, pH 7. 2, 12.5mM β- SMAD3 protein with concentration of 0.2 µg/ µl. glycerol-phosphate, 20mM MgC1 2, 5mM EGTA, 2mM EDTA. Add 0.25mM DTT to Kinase Assay Buffer prior to use.

Assay Protocol

Step 1. Thaw [33 P]-ATP Assay Cocktail in shielded container in a designated radioactive working area. Step 2. Thaw the Active EIF2AK3, Kinase Assay Buffer, Substrate and Kinase Dilution Buffer on ice. Step 3. In a pre-cooled microfuge tube, add the following reaction components bringing the initial reaction volume up to 20 µl: Component 1. 10 µl of diluted Active EIF2AK3 (Catalog #E11-11G) Component 2. 10 µl of 0.2 µg/ µl stock solution of substrate (Catalog #S12-30G)

Step 4. Set up the blank control as outlined in step 3, excluding the addition of the substrate. Replace the substrate with an equal volume of distilled H 2O. Step 5. Initiate the reaction by the addition of 5 µl [ 33 P]-ATP Assay Cocktail bringing the final volume up to 25 µl and incubate the mixture in a water bath at 30 oC for 15 minutes. Step 6. After the 15 minute incubation period, terminate the reaction by spotting 20 µl of the reaction mixture onto individual pre-cut strips of phosphocellulose P81 paper. Step 7. Air dry the pre-cut P81 strip and sequentially wash in a 1% phosphoric acid solution (dilute 10ml of phosphoric acid and make a 1L solution with distilled H2O) with constant gentle stirring. It is recommended that the strips be washed a total of 3 intervals for approximately 10 minutes each. Step 8. Count the radioactivity on the P81 paper in the presence of scintillation fluid in a scintillation counter. Step 9. Determine the corrected cpm by removing the blank control value (see Step 4) for each sample and calculate the kinase specific activity as outlined below.

Calculation of [P 33 ]-ATP Specific Activity (SA) (cpm/pmol)

Specific activit y (SA) = cpm for 5 µl [33 P] -ATP / pmoles of ATP (in 5 µl of a 250 µM ATP stock solution, i.e., 1250 pmoles)

Kinase Specific Activity (SA) (pmol/min/ µµµg or nmol/min/mg)

Corrected cpm from reaction / [(SA of 33 P-ATP in cpm/pmol)*(Reaction time in min)*( amount in µg or mg)]*[(Reaction Volume) / (Spot Volume)]

To place your order, please contact us by phone 1-(604)-232-4600, fax 1-604-232-4601 or by email: [email protected] www.signalchem.com

FOR IN VITRO RESEARCH PURPOSES ONLY. NOT INTENDED FOR USE IN HUMAN OR ANIMALS.