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181402 ASK1, GST-Fusion, Human, Recombinant, S. frugiperda

181402
  
Purchase on Sigma-Aldrich

Overview

Replacement Information
Description
Overview

This product has been discontinued.





Recombinant, human ASK1 (accession no. NM_005923), consisting of amino acids 649-946, fused at the N-terminus to GST and expressed in S. frugiperda insect cells using a baculovirus expression system. ASK1, also known as MAPKKK5, activates MKK3, MKK4 (SEK1), and MKK6, is a serine/threonine kinase that is activated in response to TNFα. It plays a central role in stress-induced apoptosis; overexpression of ASK1 induces apoptotic cell death. The active kinase is useful in the study of ASK1 activity regulation and inhibitor screening.
Catalogue Number181402
Brand Family Calbiochem®
SynonymsMEKK5, MAPKKK5
Application Data

The specific activity of ASK1 was measured as outlined above.
References
ReferencesNishitoh, H. et al.1998. Molec. Cell 2, 389.
Ichijo, H. et al. 1997. Science 275, 90.
Product Information
Unit of DefinitionKinase activity is measured as the molar amount of phosphate incorporated into MBP at 30°C using a final concentration of 50 µM ATP.
FormLiquid
FormulationIn 150 mM NaCl, 50 mM Tris-HCl, 0.25 mM DTT, 0.1 mM EGTA, 0.1 mM EDTA, 0.1 mM PMSF, 25% glycerol, pH 7.5.
Applications
Biological Information
Purity≥90% by SDS-PAGE
Specific Activity≥95 nmol/min/mg
Concentration Label Please refer to vial label for lot-specific concentration
Physicochemical Information
Dimensions
Materials Information
Toxicological Information
Safety Information according to GHS
Safety Information
Product Usage Statements
Storage and Shipping Information
Ship Code Dry Ice Only
Toxicity Standard Handling
Storage ≤ -70°C
Avoid freeze/thaw Avoid freeze/thaw
Do not freeze Ok to freeze
Special InstructionsFollowing initial thaw, aliquot and freeze (-70°C).
Packaging Information
Transport Information
Supplemental Information
Specifications

Documentation

ASK1, GST-Fusion, Human, Recombinant, S. frugiperda Certificates of Analysis

TitleLot Number
181402

References

Reference overview
Nishitoh, H. et al.1998. Molec. Cell 2, 389.
Ichijo, H. et al. 1997. Science 275, 90.
Data Sheet

Note that this data sheet is not lot-specific and is representative of the current specifications for this product. Please consult the vial label and the certificate of analysis for information on specific lots. Also note that shipping conditions may differ from storage conditions.

Revision21-October-2010 JSW
SynonymsMEKK5, MAPKKK5
Application Data

The specific activity of ASK1 was measured as outlined above.
DescriptionRecombinant, human ASK1 (accession no. NM_005923), consisting of amino acids 649-946, fused at the N-terminus to GST and expressed in S. frugiperda insect cells using a baculovirus expression system. ASK1, also known as MAPKKK5, activates MKK3, MKK4 (SEK1), and MKK6, is a serine/threonine kinase that is activated in response to TNFα. It plays a central role in stress-induced apoptosis; overexpression of ASK1 induces apoptotic cell death. The active kinase is useful in the study of ASK1 activity regulation and inhibitor screening.
FormLiquid
FormulationIn 150 mM NaCl, 50 mM Tris-HCl, 0.25 mM DTT, 0.1 mM EGTA, 0.1 mM EDTA, 0.1 mM PMSF, 25% glycerol, pH 7.5.
Concentration Label Please refer to vial label for lot-specific concentration
Recommended reaction conditions
Activity Assay Materials Required • Kinase Assay Buffer: 25 mM MOPS, 25 mM MgCl2, 12.5 mM β-glycerophosphate, 5 mM EGTA, 2 mM EDTA, pH 7.2; add 0.25 mM DTT just prior to use • Kinase Dilution Buffer: Kinase Assay Buffer diluted 1:5 with 50 ng/µl BSA • ASK1: Prepare serial dilutions using Kinase Dilution Buffer as outlined in the sample data below or as deemed necessary for individual experimental conditions • 10 mM ATP Stock Solution: Dissolve 55 mg ATP in 10 ml Kinase Assay Buffer; dispense into 200 µl aliquots and freeze (-20°C) • 250 µM [32P]-ATP Assay Cocktail: 150 µl 10 mM ATP stock solution, 100 µl [32P]-ATP (1 mCi/100 µl), 5.75 ml Kinase Assay Buffer; dispense into 1 ml aliquots and freeze (-20°C) • Substrate: Dissolve myelin basic protein (MBP) in dH2O to a final concentration of 1 mg/ml
Activity Assay Protocol 1. Thaw an aliquot of [32P]-ATP Assay Cocktail in a shielded container in a designated radioactive working area. 2. Thaw the ASK1, Kinase Assay Buffer, Substrate, and Enzyme Dilution Buffer on ice. 3. In a pre-cooled tube, add the following components: • 10 µl diluted ASK1 • 10 µl 1 mg/ml Substrate stock 4. Prepare a Blank as outlined in step 3, replacing the 10 µl Substrate with an equal volume of water. 5. Initiate the reaction by adding 5 µl [32P]-ATP Assay Cocktail (final volume is 25 µl) and incubating in a water bath at 30°C for 15 min. 6. 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 strips and sequenentially wash in 1% phosphoric acid with constant, gentle stirring for 10 min. Repeat for a total of 3 washes. 8. Count the radioactivity in presence of scintillation fluid. 9. Determine the corrected cpm by substracting the value of the Blank from each sample and calculate as follows:

Figure 1: Calculation of Activity

Purity≥90% by SDS-PAGE
Specific activity≥95 nmol/min/mg
Unit definitionKinase activity is measured as the molar amount of phosphate incorporated into MBP at 30°C using a final concentration of 50 µM ATP.
Storage ≤ -70°C
Avoid freeze/thaw
Do Not Freeze Ok to freeze
Special InstructionsFollowing initial thaw, aliquot and freeze (-70°C).
Toxicity Standard Handling
ReferencesNishitoh, H. et al.1998. Molec. Cell 2, 389.
Ichijo, H. et al. 1997. Science 275, 90.