Recombinant Human Active p38 alpha Protein, CF

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The approximate molecular weight is 67 kDa and the average purity is 90%.

Product Details

Summary
Reactivity HuSpecies Glossary
Applications Bioactivity
Format
Carrier-Free

Order Details

Recombinant Human Active p38 alpha Protein, CF Summary

Details of Functionality
The activity of p38 alpha  is typically 148-201 nmol/min/mg using an ATF2 substrate (see activity assay protocol).
Source
Spodoptera frugiperda, Sf 9 (baculovirus)-derived human p38 alpha protein
Accession # NM_139012
Accession #
N-terminal Sequence
Using an N-terminal GST tag
Protein/Peptide Type
Recombinant Enzymes
Gene
MAPK14
Purity
>70%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane

Applications/Dilutions

SDS-PAGE
67 kDa

Packaging, Storage & Formulations

Storage
This product is stable at ≤ ‑70° C for up to 1 year from the date of receipt. For optimal storage, aliquot into smaller quantities after centrifugation and store at recommended temperature. Avoid repeated freeze-thaw cycles.
Buffer
Supplied in 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.25 mM DTT, 10 mM glutathione, 0.1 mM EDTA, 0.1 mM PMSF, and 25% glycerol.
Purity
>70%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane
Assay Procedure
  • Active Kinase - Active p38 alpha (0.1 μg/μL) diluted with Kinase Dilution Buffer III as shown in Figure 2. Note: These are suggested working dilutions. Optimal dilutions should be determined by each laboratory for each application.
  • Kinase Assay Buffer I, pH 7.2 - 25 mM MOPS (pH 7.2), 12.5 mM beta -glycerolphosphate, 25 mM MgCl2, 5 mM EGTA, 2 mM EDTA. Add 0.25 mM DTT to the Kinase Assay Buffer prior to use.
    Kinase Dilution Buffer III - Kinase Assay Buffer I diluted at a 1:4 ratio (5X dilution) with 50 ng/μL BSA solution.
  • 10 mM ATP Stock Solution - Prepare the ATP Stock Solution by dissolving 55 mg of ATP in 10 mL of Kinase Assay Buffer I. Store 200 μL aliquots at ≤ -20 °C.
  • [33P]-ATP Assay Cocktail - Prepare 250 μM [33P]-ATP Assay Cocktail in a designated radioactive work area by combining 150 μL of 10 mM ATP Stock Solution, 100 μL of [33P]-ATP (1 mCi/100 μL), and 5.75 mL of Kinase Assay Buffer I. Store in 1 mL aliquots
    at ≤ -20 °C.
  • Substrate - ATF2 substrate prepared in buffer (50 mM Tris-HCl (pH 7.2), 50 mM NaCl, 5 mM EDTA, and 0.25 mM DTT) to a final concentration of 0.5 mg/mL.
  1. Thaw the [33P]-ATP Assay Cocktail in a shielded container in a designated radioactive work area.
  2. Thaw the Active p38 alpha, Kinase Assay Buffer I, Substrate, and Kinase Dilution Buffer III on ice.
  3. In a pre-cooled microfuge tube, add the following reaction components bringing the initial reaction volume up to 20 μL.
    a. Diluted Active p38 alpha : 10 μL
    b. ATF2 Substrate (0.5 mg/mL Stock Solution): 5 μL
  4. Set up the blank control as outlined in step 3, excluding the addition of the substrate. Replace the substrate with an equal volume of distilled or deionized water.
  5. Initiate the reaction by the addition of 5 μL [33P]-ATP Assay Cocktail, bringing the final volume up to 25 μL. Incubate the mixture in a water bath at 30 °C for 15 minutes.
  6. After the 15 minute incubation period, terminate the reaction by spotting 20 μL of the reaction mixture onto individual pre-cut strips of phosphocellulose P81 paper.
  7. Air dry the pre-cut P81 strip and sequentially wash in a 1% phosphoric acid solution (add 10 mL of phosphoric acid to 990 mL of distilled or deionized water) with constant gentle stirring. It is recommended that the strips be washed a total of three times for approximately 10 minutes each.
  8. Count the radioactivity on the P81 paper in the presence of scintillation fluid in a scintillation counter.
  9. Determine the corrected cpm by subtracting the blank control value (see step 4) for each sample and calculate the kinase specific activity as outlined below:


    Calculation of [33P]-ATP Specific Activity (SA) (cpm/pmol)
    Specific Activity (SA) = cpm for 5 μL [33P]-ATP/pmol of ATP (in 5 μL of a 250 μM ATP stock solution; i.e. 1250 pmol)

    Calculation of Kinase Specific Activity (SA) (pmol/minute/μg or nmol/minute/mg)
    Corrected cpm from reaction / [(SA of 33P-ATP in cpm/pmol) x (Reaction time in minutes) x (Enzyme amount in μg or mg)] x [(Reaction volume) / (Spot Volume)]

Notes

This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.

Alternate Names for Recombinant Human Active p38 alpha Protein, CF

  • MAPK14
  • p38 alpha
  • SAPK2a

Background

p38 alpha (SAPK2A) is a member of the p38 MAPK family which are activated by various environmental stresses and pro-inflammatory cytokines (1). The activation of p38 requires its phosphorylation by MAP kinase kinases (MKKs) or its autophosphorylation triggered by the interaction of MAP3K7IP1/TAB1 protein with this kinase (2). The substrates of p38 include transcription regulator ATF2, MEF2C, MAX, cell cycle regulator CDC25B, and tumor suppressor p53, which suggest the roles of this kinase in stress related transcription and cell cycle regulation, as well as in genotoxic stress response.

  1. Han, J. et al. (1994) Science 265:808.
  2. Ge, B. et al. (2002) Science 295:1291.

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Bioinformatics

Gene Symbol MAPK14
Entrez
Uniprot