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EIF2AK1 (HRI), Active (SRP5321)

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EIF2AK1 (HRI), Active (SRP5321) EIF2AK1 (HRI), active, GST-tagged, human Ò PRECISIO Kinase recombinant, expressed in Sf9 cells Catalog Number SRP5321 Storage Temperature –70 °C Synonyms: HCR, HRI, KIAA1369 Figure 1. SDS-PAGE Gel of Typical Lot: Product Description ³70% (SDS-PAGE, densitometry) EIF2AK1 (HRI) or eukaryotic translation initiation factor 2-alpha kinase 1 acts at the level of translation initiation to downregulate protein synthesis in response to stress. EIF2AK1 (HRI) can be inactivated by hemin and is activated by heme deficiency and other stimuli. It is a major protein kinase that phosphorylates EIF2 a.1 EIF2AK1 (HRI) is downregulated in the majority of ovarian cancers compared with normal ovarian tissues. EIF2AK1 (HRI) functions in iron homeostasis and may play a role in hemolytic and inflammatory anemia.2 Recombinant full-length human EIF2AK1 (HRI) was Figure 2. expressed by baculovirus in Sf9 insect cells using an Specific Activity of Typical Lot: N-terminal GST-tag. The gene accession number is 9.6–14.4 nmole/min/mg NM_014413. It is supplied in 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 10 mM glutathione, 0.1 mM EDTA, 0.25 mM DTT, 0.1 mM PMSF, and 25% glycerol. Molecular mass: ~120 kDa Precautions and Disclaimer This product is for R&D use only, not for drug, household, or other uses. Please consult the Material Safety Data Sheet for information regarding hazards and safe handling practices. Storage/Stability The product ships on dry ice and storage at –70 °C is Procedure recommended. After opening, aliquot into smaller Preparation Instructions quantities and store at –70 °C. Avoid repeated handling Kinase Assay Buffer – 25 mM MOPS, pH 7. 2, 12.5 mM and multiple freeze/thaw cycles. glycerol 2-phosphate, 25 mM MgC12, 5 mM EGTA, and 2 mM EDTA. Just prior to use, add DTT to a final concentration of 0.25 mM. Kinase Dilution Buffer – Dilute the Kinase Assay Buffer 5-fold with a 50 ng/mL BSA solution. Kinase Solution – Dilute the active EIF2AK1 (HRI) 6. Air dry the precut P81 strip and sequentially wash (0.1 mg/mL) with Kinase Dilution Buffer to the desired in the 1% phosphoric acid solution with constant concentration. gentle stirring. It is recommended the strips be Note: The specific activity plot may be used as a washed a total of 3 times of ~10 minutes each. guideline (see Figure 2). It is recommended the 7. Set up a radioactive control to measure the total researcher perform a serial dilution of active EIF2AK1 g-33P-ATP counts introduced into the reaction. Spot (HRI) kinase for optimal results. 5 mL of the g-33P-ATP Assay Cocktail on a precut P81 strip. Dry the sample for 2 minutes and read 10 mM ATP Stock Solution – Dissolve 55 mg of ATP in the counts. Do not wash this sample. 10 mL of Kinase Assay Buffer. Store in 200 mL aliquots 8. Count the radioactivity on the P81 paper in the at –20 °C. presence of scintillation fluid in a scintillation counter. g-33P-ATP Assay Cocktail (250 mM) – Combine 5.75 mL 9. Determine the corrected cpm by subtracting the of Kinase Assay Buffer, 150 mL of 10 mM ATP Stock blank control value (see step 3) from each sample Solution, 100 mL of g-33P-ATP (1 mCi/100 mL). Store in and calculate the kinase specific activity 1 mL aliquots at –20 °C. Calculations: Substrate Solution – RS Repeat Peptide substrate 1. Specific Radioactivity (SR) of ATP (cpm/nmole) (GRSRSRSRSRSRSRSR) diluted in distilled water to a 33 final concentration of 1 mg/mL. SR = cpm of 5 mL of g- P-ATP Assay Cocktail nmole of ATP 1% phosphoric acid solution – Dilute 10 mL of concentrated phosphoric acid to a final volume of 1 L cpm – value from control (step 7) with water. nmole – 1.25 nmole (5 mL of 250 mM ATP Assay Cocktail) Kinase Assay This assay involves the use of the 33P radioisotope. All 2. Specific Kinase Activity (SA) (nmole/min/mg) institutional guidelines regarding the use of radioisotopes should be followed. nmole/min/mg = Dcpm ´ (25/20) SR ´ E ´ T 1. Thaw the active EIF2AK1 (HRI), Kinase Assay Buffer, Substrate Solution, and Kinase Dilution SR = specific radioactivity of the ATP (cpm/nmole ATP) Buffer on ice. The g-33P-ATP Assay Cocktail may Dcpm = cpm of the sample – cpm of the blank (step 3) be thawed at room temperature. 25 = total reaction volume 2. In a pre-cooled microcentrifuge tube, add the 20 = spot volume following solutions to a volume of 20 mL: T = reaction time (minutes) 10 mL of Kinase Solution E = amount of enzyme (mg) 5 mL of Substrate Solution 5 mL of cold water (4 °C) References 3. Set up a blank control as outlined in step 2, 1. Hwang et al., Cloning of hHRI, human heme- substituting 5 mL of cold water (4 °C) for the regulated eukaryotic initiation factor 2-alpha kinase: Substrate Solution. down-regulated in epithelial ovarian cancers. Molec. Cells, 10, 584-591 (2000). 4. Initiate each reaction with the addition of 5 mL of the 33 2. Liu, S. et al., The function of heme-regulated elF2- g- P-ATP Assay Cocktail, bringing the final alpha kinase in murine iron homeostasis and reaction volume to 25 L. Incubate the mixture in a m macrophage maturation. J. Clin. Invest., 117, 3296- water bath at 30 °C for 15 minutes. 3305 (2007). 5. After the 15 minute incubation, stop the reaction by spotting 20 mL of the reaction mixture onto an PRECISIO is a registered trademark of Sigma-Aldrich individually precut strip of phosphocellulose P81 Co. LLC. paper. RC,MAM 12/12-1 Ó2012 Sigma-Aldrich Co. LLC. All rights reserved. SIGMA-ALDRICH is a trademark of Sigma-Aldrich Co. LLC, registered in the US and other countries. Sigma brand products are sold through Sigma-Aldrich, Inc. Purchaser must determine the suitability of the product(s) for their particular use. Additional terms and conditions may apply. Please see product information on the Sigma-Aldrich website at www.sigmaaldrich.com and/or on the reverse side of the invoice or packing slip..
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