MYLK2, active, His-tagged, human PRECISIOÒ recombinant, expressed in Sf9 cells

Catalog Number M0949 Lot Number 041M0980 Storage Temperature –70 °C

Synonyms: skMLCK, KMLC, MLCK, MLCK2 Figure 1. SDS-PAGE Gel of Lot Number 041M0980: Product Description >75% (densitometry) MYLK2 is a member of the light chain kinase family and is a calcium/ dependent exclusively expressed in adult skeletal muscle.1 MYLK2 has been proposed to participate in signaling pathways (calcium signaling pathway, focal adhesion, and regulation of ) and cellular processes (neuromuscular synaptic transmission and protein/ amino acid phosphorylation). MYLK2 is involved in multiple molecular functions as a result of various subdomains that participate in ATP binding, calmodulin binding, nucleotide binding, protein serine/threonine kinase activity, and activity.2 Figure 2. This recombinant product was expressed by Specific Activity of Lot Number 041M0980: baculovirus in Sf9 insect cells using an N-terminal 270 nmole/min/mg His-tag. The accession number is NM 033118. It is supplied in 50 mM sodium phosphate, pH 7.0, with 300 mM NaCl, 150 mM imidazole, 0.2 mM DTT, 0.1 mM PMSF, and 25% glycerol.

Molecular mass: ~74 kDa

Purity: ³70% (SDS-PAGE, see Figure 1)

Specific Activity: 229–311 nmole/min/mg (see Figure 2)

Precautions and Disclaimer This product is for R&D use only, not for drug, household, or other uses. Please consult the Material Procedure Safety Data Sheet for information regarding hazards Preparation Instructions and safe handling practices. Kinase Assay Buffer – 25 mM MOPS, pH 7.2, 12.5 mM glycerol 2-phosphate, 25 mM MgCl2, 5 mM EGTA, and Storage/Stability 2 mM EDTA. Just prior to use, add DTT to a final The product ships on dry ice and storage at –70 °C is concentration of 0.25 mM. recommended. After opening, aliquot into smaller quantities and store at –70 °C. Avoid repeated handling Kinase Dilution Buffer – Dilute the Kinase Assay Buffer and multiple freeze/thaw cycles. 5-fold with a 50 ng/ml BSA solution. Kinase Solution – Dilute the active MYLK2 (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 the washed a total of 3 times of ~10 minutes each. researcher perform a serial dilution of active MYLK2 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 – Prepare LC20 protein substrate at Calculations: a final concentration of 1.0 mg/ml. 1. Specific Radioactivity (SR) of ATP (cpm/nmole)

32 1% phosphoric acid solution – Dilute 10 ml of SR = cpm of 5 ml of g- P-ATP Assay Cocktail concentrated phosphoric acid to a final volume of 1 L nmole of ATP with water. cpm – value from control (step 7) Kinase Assay nmole – 1.25 nmole (5 ml of 250 mM ATP This assay involves the use of the 32P radioisotope. All Assay Cocktail) institutional guidelines regarding the use of radioisotopes should be followed. 2. Specific Kinase Activity (SA) (nmole/min/mg)

1. Thaw the active MYLK2, Kinase Assay Buffer, nmole/min/mg = Dcpm ´ (25/20) Substrate Solution, and Kinase Dilution Buffer on SR ´ E ´ T ice. The g-32P-ATP Assay Cocktail may be thawed at room temperature. SR = specific radioactivity of the ATP (cpm/nmole ATP) 2. In a pre-cooled microcentrifuge tube, add the Dcpm = cpm of the sample – cpm of the blank (step 3) following solutions to a volume of 20 ml: 25 = total reaction volume 10 ml of Kinase Solution 20 = spot volume 5 ml of Substrate Solution T = reaction time (minutes) E = amount of enzyme (mg) 2.5 ml of 5 mM CaCl2 solution containing 0.75 mg Calmodulin 2.5 ml of water References 3. Set up a blank control as outlined in step 2, 1. Soung, Y.H. et al., Mutational analysis of the kinase domain of MYLK2 gene in common human substituting 5 ml of cold water (4 °C) for the cancers. Pathol. Res. Pract., 202, 137-140 (2006). Substrate Solution. 2. Toth-Zsamboki, E. et al., P2X1-mediated ERK2 4. Initiate each reaction with the addition of 5 ml of the 32 activation amplifies the collagen-induced platelet g- P-ATP Assay Cocktail, bringing the final secretion by enhancing myosin light chain kinase reaction volume to 25 ml. Incubate the mixture in a activation. J. Biol. Chem., 278, 46661-46667 water bath at 30 °C for 15 minutes. (2003). 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-AldrichÒ individually precut strip of phosphocellulose P81 Biotechnology LP and Sigma-Aldrich Co. paper. RC,MAM 05/11-1

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.