HIPK2, active, GST-tagged, mouse Ò PRECISIO Kinase recombinant, expressed in Sf9 cells

Catalog Number SRP5313 Storage Temperature –70 °C

Synonyms: Stank, 1110014O20Rik, B230339E18Rik Figure 1. SDS-PAGE Gel of Typical Lot: Product Description ³70% (SDS-PAGE, densitometry) HIPK2 is a conserved / nuclear kinase that interacts with homeodomain transcription factors. HIPK2 enhances expression of target , resulting in growth arrest and the enhancement of UV-induced that could be inhibited by antisense to HIPK2.1 HIPK2 is a target for mediated ubiquitin-dependent degradation that only occurs in growth-arresting conditions when MDM2 was efficiently induced by p53.2 The knockdown of HIPK2 using small interfering RNA reduced p53 binding and activation of p53R2 resulting in impaired UV-induced DNA repair. Figure 2. Specific Activity of Typical Lot: Recombinant mouse HIPK2 (153-564) was expressed 88–132 nmole/min/mg by baculovirus in Sf9 insect cells using an N-terminal GST-tag. The accession number is BC031904. 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: ~72 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. Procedure Storage/Stability Preparation Instructions Kinase Assay Buffer – 25 mM MOPS, pH 7. 2, 12.5 mM The product ships on dry ice and storage at –70 °C is glycerol 2-phosphate, 25 mM MgC1 , 5 mM EGTA, and recommended. After opening, aliquot into smaller 2 2 mM EDTA. Just prior to use, add DTT to a final quantities and store at –70 °C. Avoid repeated handling concentration of 0.25 mM. and multiple freeze/thaw cycles. Kinase Dilution Buffer – Dilute the Kinase Assay Buffer 5-fold with a 50 ng/ml BSA solution. Kinase Solution – Dilute the active HIPK2 (0.1 mg/mL) 6. Air dry the precut P81 strip and sequentially wash with Kinase Dilution Buffer to the desired concentration. in the 1% phosphoric acid solution with constant Note: The specific activity plot may be used as a gentle stirring. It is recommended the strips be guideline (see Figure 2). It is recommended the washed a total of 3 times of ~10 minutes each. researcher perform a serial dilution of active HIPK2 7. Set up a radioactive control to measure the total kinase for optimal results. g-33P-ATP counts introduced into the reaction. Spot 5 mL of the g-33P-ATP Assay Cocktail on a precut 10 mM ATP Stock Solution – Dissolve 55 mg of ATP in P81 strip. Dry the sample for 2 minutes and read 10 mL of Kinase Assay Buffer. Store in 200 mL aliquots the counts. Do not wash this sample. at –20 °C. 8. Count the radioactivity on the P81 paper in the presence of scintillation fluid in a scintillation g-33P-ATP Assay Cocktail (250 mM) – Combine 5.75 mL counter. of Kinase Assay Buffer, 150 mL of 10 mM ATP Stock 9. Determine the corrected cpm by subtracting the Solution, 100 mL of g-33P-ATP (1 mCi/100 mL). Store in blank control value (see step 3) from each sample 1 mL aliquots at –20 °C. and calculate the kinase specific activity

Substrate Solution – MBP Protein peptide substrate Calculations: diluted in distilled water to a final concentration of 1. Specific Radioactivity (SR) of ATP (cpm/nmole) 1 mg/mL. SR = cpm of 5 mL of g-33P-ATP Assay Cocktail 1% phosphoric acid solution – Dilute 10 mL of nmole of ATP concentrated phosphoric acid to a final volume of 1 L with water. cpm – value from control (step 7) nmole – 1.25 nmole (5 mL of 250 mM ATP Kinase Assay Assay Cocktail) This assay involves the use of the 33P radioisotope. All institutional guidelines regarding the use of 2. Specific Kinase Activity (SA) (nmole/min/mg) radioisotopes should be followed. nmole/min/mg = Dcpm ´ (25/20) 1. Thaw the active HIPK2, Kinase Assay Buffer, SR ´ E ´ T Substrate Solution, and Kinase Dilution Buffer on ice. The g-33P-ATP Assay Cocktail may be thawed SR = specific radioactivity of the ATP (cpm/nmole ATP) at room temperature. Dcpm = cpm of the sample – cpm of the blank (step 3) 2. In a pre-cooled microcentrifuge tube, add the 25 = total reaction volume following solutions to a volume of 20 mL: 20 = spot volume 10 mL of Kinase Solution T = reaction time (minutes) 5 mL of Substrate Solution E = amount of (mg) 5 mL of cold water (4 °C) 3. Set up a blank control as outlined in step 2, References substituting 5 mL of cold water (4 °C) for the 1. Hofmann, T.G. et al., Regulation of p53 activity by Substrate Solution. its interaction with homeodomain-interacting -2. Nature Cell Biol., 4, 1-10 (2002). 4. Initiate each reaction with the addition of 5 mL of the 33 2. Rinaldo, C. et al., MDM2-regulated degradation of g- P-ATP Assay Cocktail, bringing the final HIPK2 prevents p53 Ser46 and reaction volume to 25 L. Incubate the mixture in a m DNA damage-induced apoptosis. Molec. Cell, 25, water bath at 30 °C for 15 minutes. 739-750 (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

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