CAMK1 Active mouse, recombinant GST-tagged, expressed in E. coli cells

Catalog Number C7245 Lot Number 019K1564 Storage Temperature –70 °C

Synonyms: AI505105; D6Ertd263e Figure 1. SDS-PAGE Gel of Lot Number 019K1564: Product Description >90% (densitometry) CAMK1 is a serine/threonine protein that is a 250 member of the multifunctional calcium/- 150 dependent family. CAMK1 is ubiquitously 100 75 expressed and phosphorylates a number of proteins CAMK1 including SYN1, SYN2, CREB, and CFTR. In addition, 50 the Numb family of proteins may also be intracellular 37 targets for CAMK1 and they may also be regulated by phosphorylation-dependent interaction with 14-3-3 25 protein.1 CAMK1 also plays an important role in the trafficking of HDAC7 between the cytoplasm and the nucleus. CAMK1 phosphorylates HDAC7 on multiple Figure 2. sites, which leads to alteration in localization of Specific Activity of Lot Number 019K1564: HDAC7.2 3,214 nmole/min/mg

This recombinant product was expressed in E. coli cells using an N-terminal GST tag. The accession number is NM 133926. It is supplied in 50 mM Tris-HCl, 1,400,000 pH 7.5, with 150 mM NaCl, 0.25 mM DTT, 0.1 mM 1,050,0001,400,000 EGTA, 0.1 mM EDTA, 0.1 mM PMSF, and 25% 700,0001,050,000 glycerol. 350,000700,000 350,0000 Activity (cpm) Molecular mass: ~70 kDa 0 6 12 18 24 Activity (cpm) 0 6Protein12 (ng) 18 24 Purity: ³70% (SDS-PAGE, see Figure 1)

Specific Activity: 2,732–3,696 nmole/min/mg (see Procedure Figure 2) Preparation Instructions Kinase Assay Buffer – 25 mM MOPS, pH 7.2, 12.5 mM Precautions and Disclaimer glycerol 2-phosphate, 25 mM MgCl2, 5 mM EGTA, and This product is for R&D use only, not for drug, 2 mM EDTA. Just prior to use, add DTT to a final household, or other uses. Please consult the Material concentration of 0.25 mM. Safety Data Sheet for information regarding hazards and safe handling practices. Kinase Dilution Buffer – Dilute the Kinase Assay Buffer 5-fold with a 50 ng/ml BSA solution. Storage/Stability The product ships on dry ice and storage at –70 °C is recommended. After opening, aliquot into smaller quantities and store at –70 °C. Avoid repeated handling and multiple freeze/thaw cycles. Kinase Solution – Dilute the Active CAMK1 (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 7. Set up a radioactive control to measure the total CAMK1 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 (KKALRRQETVDAL-amide) 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 CAMK1, 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 (mg) 7.5 ml of Substrate Solution References 2.5 ml of 5 mM CaCl2 solution containing 0.75 mg Calmodulin 1. Tokumitsu, H. et al., Phosphorylation of Numb family proteins. Possible involvement of 3. Set up a blank control as outlined in step 2, 2+ substituting 7.5 ml of cold water (4 °C) for the Ca /calmodulin-dependent protein . J. Biol. Substrate Solution. Chem., 280, 35108-35118 (2005). 2. Gao, C. et al., CRM1 mediates nuclear export of 4. Initiate each reaction with the addition of 5 ml of the HDAC7 independently of HDAC7 phosphorylation -32P-ATP Assay Cocktail, bringing the final g and association with 14-3-3s. FEBS Lett., 580, reaction volume to 25 ml. Incubate the mixture in a 5096-5104 (2006). water bath at 30 °C for 15 minutes. TLD,MAM 02/09-1 5. After the 15 minute incubation, stop the reaction by spotting 20 ml of the reaction mixture onto an 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.