P38α substrate peptide


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P38α substrate peptide PE-210 1 mg 183,60 Add to Basket/Quote Add to Notepad

For in vitro use only!

Shipping: shipped on blue ice

Storage Conditions: store at -20 °C
avoid freeze/thaw cycles

Shelf Life: 12 months


Molecular Weight: 2048 g/mol

Purity: > 95 % (HPLC)

Form: lyophilized (reconstitution of 1 mg in 245 μl ddH2O results in a 2 mM solution used in the activity assay)

The synthetic peptide RRRLVEPLTPSGEAPNQK
can be used as a substrate for p38alpha kinase in in vitro kinase assays. Its sequence is derived from epidermal growth factor receptor (amino acids 643-656 of accession number AAH94761

In vitro Kinase Assay

Assay Components
Assay buffer (AB): 50 mM Tris-HCl, pH 7.5, 0.1 mM EGTA, and 10 mM DTT.
Enzyme dilution buffer: AB with 1 mg/ml BSA
Substrate: p38alpha peptide substrate RRRLVEPLTPSGEAPNQK, 2 mM
Protein kinase: p38alpha, 1.25 - 10 ng/ microliter diluted in EDB directly before use
Magnesium/ATP Cocktail: 80 mM MgCl2, 4 microM ATP
Diluted [gamma-32P]ATP: Mix 197 microliter Magnesium/ATP cocktail with 3 microliter (30
microCi) [gamma-32P]ATP (3,000 Ci/mmol, e.g. from Hartmann Analytic, Braunschweig)

Assay procedure
All compounds are pipetted into a microcentrifuge tube on ice
1. Add 10 microliter AB
2. Add 10 microliter peptide substrate
3. Add 2.5 microliter p38alpha (12.5 - 100 ng/assay)
4. Add 10 microliter of the diluted [gamma-32P]ATP
5. Incubate 10 min at 30 oC.
6. Stop the reaction by setting samples on ice
7. Remove 20 microliter and spot on P81 paper (let bind to the paper for 30 sec)
8. Immerse the paper in 0.75% phosphoric acid, gently shake on a rotator
9. Wash 3 x with phosphoric acid
10. Wash 1 x with acetone
11. Dry under infrared light
12. Read in scintillation counter or Instant Imager