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STK3 Active (S6573)

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STK3 Active (S6573) STK3 Active human, recombinant GST-tagged, expressed in Sf9 cells Catalog Number S6573 Lot Number 019K1598 Storage Temperature –70 °C Synonyms: KRS1; MST2 Figure 1. SDS-PAGE Gel of Lot Number 019K1598: Product Description >80% (densitometry) STK3, also known as MST2, encodes a protein of 491 amino acids which contains an N-terminal catalytic 170 1 130 domain characteristic of STKs. STK3 and STK4 share 100 STK3 94% amino acid identity in the catalytic domain and 72 78% identity overall. RAF1 has been shown to counteract apoptosis by suppressing the activation of 55 mammalian sterile 20-like kinase (MST2). STK3 is a 40 kinase that is activated by the proapoptotic agents, 33 straurosporine and FAS ligand.2 STK3 activation presumably allows cells to resist unfavorable environmental conditions. Figure 2. This recombinant product was expressed by Specific Activity of Lot Number 019K1598: baculovirus in Sf9 insect cells using an N-terminal 277 nmole/min/mg GST-tag. The gene accession number is BC 010640. It is supplied in 50 mM Tris-HCl, pH 7.5, with 150 mM 500,000 NaCl, 0.25 mM DTT, 0.1 mM EGTA, 0.1 mM EDTA, 0.1 mM PMSF, and 25% glycerol. 400,000 300,000 Molecular mass: ~87 kDa ctivity (cpm) 200,000 A 100,000 Purity: ³70% (SDS-PAGE, see Figure 1) 0 0 40 80 120 160 Specific Activity: 235–319 nmole/min/mg (see Figure 2) Protein (ng) Precautions and Disclaimer This product is for R&D use only, not for drug, Procedure household, or other uses. Please consult the Material Preparation Instructions Safety Data Sheet for information regarding hazards Kinase Assay Buffer – 25 mM MOPS, pH 7.2, 12.5 mM and safe handling practices. glycerol 2-phosphate, 25 mM MgCl2, 5 mM EGTA, and 2 mM EDTA. Just prior to use, add DTT to a final Storage/Stability concentration of 0.25 mM. The product ships on dry ice and storage at –70 °C is recommended. After opening, aliquot into smaller Kinase Dilution Buffer – Dilute the Kinase Assay Buffer quantities and store at –70 °C. Avoid repeated handling 5-fold with a 50 ng/ml BSA solution. and multiple freeze/thaw cycles. Kinase Solution – Dilute the Active STK3 (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 lot-specific specific activity plot may be used gentle stirring. It is recommended the strips be as a guideline (see Figure 2). It is recommended that washed a total of 3 times of ~10 minutes each. the researcher perform a serial dilution of Active STK3 7. Set up a radioactive control to measure the total kinase for optimal results. g-32P-ATP counts introduced into the reaction. Spot 5 ml of the g-32P-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 at the counts. Do not wash this sample. –20 °C. 8. Count the radioactivity on the P81 paper in the presence of scintillation fluid in a scintillation g-32P-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-32P-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 – Dissolve the synthetic peptide Calculations: substrate myelin basic protein (MBP) in water at a final 1. Specific Radioactivity (SR) of ATP (cpm/nmole) concentration of 1 mg/ml. SR = cpm of 5 ml of g-32P-ATP Assay Cocktail 1% phosphoric acid solution – Dilute 10 ml of nmole of ATP 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 32P 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 STK3, Kinase Assay Buffer, Substrate Solution, and Kinase Dilution Buffer on SR = specific radioactivity of the ATP (cpm/nmole ATP) ice. The g-32P-ATP Assay Cocktail may be thawed Dcpm = cpm of the sample – cpm of the blank (step 3) 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) 10 ml of Substrate Solution 3. Set up a blank control as outlined in step 2, References substituting 10 ml of cold water (4 °C) for the 1. Creasy, C.L. et al., Cloning and characterization of Substrate Solution. a member of the MST subfamily of Ste20-like 4. Initiate each reaction with the addition of 5 ml of the kinases. Gene, 167, 303-306 (1995). g-32P-ATP Assay Cocktail, bringing the final 2. Lee, K.K. et al., MST, a physiological caspase substrate, highly sensitizes apoptosis both reaction volume to 25 ml. Incubate the mixture in a upstream and downstream of caspase activation. J. water bath at 30 °C for 15 minutes. Biol. Chem., 276, 19276-19285 (2001). 5. After the 15 minute incubation, stop the reaction by spotting 20 ml of the reaction mixture onto an TLD,MAM 02/09-1 individually precut strip of phosphocellulose P81 paper. Sigma brand products are sold through Sigma-Aldrich, Inc. Sigma-Aldrich, Inc. warrants that its products conform to the information contained in this and other Sigma-Aldrich publications. Purchaser must determine the suitability of the product(s) for their particular use. Additional terms and conditions may apply. Please see reverse side of the invoice or packing slip..
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