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MAPKAPK3 Active Human, Recombinant GST-Tagged, Expressed in Sf9 Cells

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MAPKAPK3 Active Human, Recombinant GST-Tagged, Expressed in Sf9 Cells MAPKAPK3 Active human, recombinant GST-tagged, expressed in Sf9 cells Catalog Number M8322 Lot Number 039K0769 Storage Temperature –70 °C Synonyms: 3PK; 3pK; MAPKAP3 Figure 1. SDS-PAGE Gel of Lot Number 039K0769: Product Description >90% (densitometry) MAPKAPK3 is closely related to MAPKAPK2 sharing 1 72% nucleotide and 75% amino acid identity. 170 MAPKAPK3 is activated by growth inducers and stress 130 stimulation of cells. In vitro studies demonstrated ERK, 100 p38 MAP kinase, and Jun N-terminal kinase were all 72 MAPKAPK3 able to phosphorylate and activate this kinase, which 55 suggested the role of MAPKAPK3 as an integrative 40 element of signaling in both mitogen and stress 2 responses. 33 This recombinant product was expressed by baculovirus in Sf9 insect cells using an N-terminal Figure 2. GST-tag. The gene accession number is NM 004635. It Specific Activity of Lot Number 039K0769: is supplied in 50 mM Tris-HCl, pH 7.5, with 150 mM 636 nmole/min/mg NaCl, 0.25 mM DTT, 0.1 mM EGTA, 0.1 mM EDTA, 0.1 mM PMSF, and 25% glycerol. 1,000,000 Molecular mass: ~69 kDa 800,000 600,000 Purity: ³70% (SDS-PAGE, see Figure 1) 400,000 Specific Activity: 541–731 nmole/min/mg (see Figure 2) Activity (cpm) 200,000 0 Precautions and Disclaimer 0 40 80 120 160 This product is for R&D use only, not for drug, Protein (ng) household, or other uses. Please consult the Material Safety Data Sheet for information regarding hazards and safe handling practices. Procedure Preparation Instructions Storage/Stability Kinase Assay Buffer – 25 mM MOPS, pH 7.2, 12.5 mM glycerol 2-phosphate, 25 mM MgCl , 5 mM EGTA, and The product ships on dry ice and storage at –70 °C is 2 2 mM EDTA. Just prior to use, add DTT to a final recommended. After opening, aliquot into smaller concentration of 0.25 mM. quantities and store at –70 °C. Avoid repeated handling 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 MAPKAPK3 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 lot-specific specific activity plot may be used washed a total of 3 times of ~10 minutes each. as a guideline (see Figure 2). It is recommended that 7. Set up a radioactive control to measure the total the researcher perform a serial dilution of Active g-32P-ATP counts introduced into the reaction. Spot MAPKAPK3 kinase for optimal results. 5 ml of the g-32P-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 at 8. Count the radioactivity on the P81 paper in the –20 °C. presence of scintillation fluid in a scintillation counter. g-32P-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-32P-ATP (1 mCi/100 ml). Store in and calculate the kinase specific activity 1 ml aliquots at –20 °C. Calculations: Substrate Solution – Dissolve the synthetic peptide 1. Specific Radioactivity (SR) of ATP (cpm/nmole) substrate (RRLNRQLSVA-amide) in water at a final 32 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 cpm – value from control (step 7) concentrated phosphoric acid to a final volume of 1 L nmole – 1.25 nmole (5 ml of 250 mM ATP with water. Assay Cocktail) Kinase Assay 2. Specific Kinase Activity (SA) (nmole/min/mg) This assay involves the use of the 32P radioisotope. All institutional guidelines regarding the use of nmole/min/mg = Dcpm ´ (25/20) radioisotopes should be followed. SR ´ E ´ T 1. Thaw the Active MAPKAPK3, Kinase Assay Buffer, SR = specific radioactivity of the ATP (cpm/nmole ATP) Substrate Solution, and Kinase Dilution Buffer on Dcpm = cpm of the sample – cpm of the blank (step 3) ice. The g-32P-ATP Assay Cocktail may be thawed 25 = total reaction volume at room temperature. 20 = spot volume 2. In a pre-cooled microcentrifuge tube, add the T = reaction time (minutes) following solutions to a volume of 20 ml: E = amount of enzyme (mg) 10 ml of Kinase Solution 10 ml of Substrate Solution References 3. Set up a blank control as outlined in step 2, 1. Sithanandam, G. et al., 3pK a new mitogen- substituting 10 ml of cold water (4 °C) for the activated protein kinase-activated protein kinase Substrate Solution. located in the small cell lung cancer tumor 4. Initiate each reaction with the addition of 5 ml of the suppressor gene region. Molec. Cell. Biol., 16, 868- g-32P-ATP Assay Cocktail, bringing the final 876 (1996). 2. Ludwig, S. et al., 3pK, a novel mitogen-activated reaction volume to 25 ml. Incubate the mixture in a protein (MAP) kinase-activated protein kinase, is water bath at 30 °C for 15 minutes. targeted by three MAP kinase pathways. Molec. 5. After the 15 minute incubation, stop the reaction by Cell. Biol., 16, 6687-6697 (1996). spotting 20 ml of the reaction mixture onto an individually precut strip of phosphocellulose P81 TLD,MAM 04/09-1 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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