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539672
Sigma-AldrichPKCδ, Human, Recombinant, S. frugiperda
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Description
Overview
Full-length, recombinant, human PKCδ expressed in S. frugiperda insect cells using a baculovirus expression system. Suitable for studies of PKCδ and its regulation. PKCδ is a Ca2+-independent and phospholipid-dependent enzyme.
Catalogue Number
539672
Brand Family
Calbiochem®
Synonyms
Protein Kinase Cδ
References
References
Hug, J. and Sarre, T.F. 1993. Biochem. J. 291, 329. Kazanietz, M.G., et al. 1993. Mol.Pharmacol. 44, 298. Koide, H., et al. 1992. Proc. Natl. Acad. Sci. USA 89, 1149.
Product Information
Unit of Definition
One unit is defined as the amount of enzyme that will transfer 1 nmol of phosphate to PKC<sub>ε</sub> Peptide Substrate (<a href ="/Products/ProductDisplay.asp→catNO=539562">Cat. No. 539562</a>) per min at 30°C, pH 7.5.
Form
Liquid
Formulation
In 250 mM NaCl, 20 mM HEPES, 5 mM DTT, 2 mM EDTA, 2 mM EGTA, 50% glycerol, 0.05% TRITON® X-100 Detergent, pH 7.5.
PKCδ, Human, Recombinant, S. frugiperda Certificates of Analysis
Title
Lot Number
539672
References
Reference overview
Hug, J. and Sarre, T.F. 1993. Biochem. J. 291, 329. Kazanietz, M.G., et al. 1993. Mol.Pharmacol. 44, 298. Koide, H., et al. 1992. Proc. Natl. Acad. Sci. USA 89, 1149.
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.
Revision
08-September-2008 RFH
Synonyms
Protein Kinase Cδ
Description
Full-length, recombinant, human PKCδ expressed in S. frugiperda insect cells using a baculovirus expression system. PKC δ is classified as a novel PKC isozyme because it does not exhibit calcium dependence and it is activated in vitro by phorbol esters. Suitable for studies of PKCδ and its regulation. PKCδ is a Ca2+-independent and phospholipid-dependent enzyme.
Form
Liquid
Formulation
In 250 mM NaCl, 20 mM HEPES, 5 mM DTT, 2 mM EDTA, 2 mM EGTA, 50% glycerol, 0.05% TRITON® X-100 Detergent, pH 7.5.
Concentration Label
Please refer to vial label for lot-specific concentration
Recommended reaction conditions
Protocol for Recombinant PKC δNote: this preparation is purified to near homogeneity, so it may behave differently than crude preparations. In particular, the concentration and quality of the phospholipids used does not seem to be important.
Lipid Mixture:
1. Determine the total amount of lipid mixture needed. Each reaction requires 20 µg of phosphatidylserine (1.2 µl of 10 mg/ml phosphatidylserine in CHCl3) and 2 mg of diacylglycerol (0.6 µl of 2 mg/ml diacylglycerol in CHCl3). Preparing 10% more lipid mixture than required is recommended to account for losses while pipetting.
2. Using a Hamilton syringe that has been cleaned with methanol, transfer the required volume of each lipid stock solution to a 12 x 75 mm glass tube.
3. Evaporate off the chloroform.
4. Resuspend in 10 µl of 10 mM HEPES, pH 7.4, 0.3% TRITON® X-100 detergent.
5. Vortex for at least 2 min.
6. Place the lipid mixture in a 40°C water bath for 5 min prior to adding the reaction mixture.
Reaction Mixture:
2.4 µl 500 mM HEPES, pH 7.4
6 µl 100 mM MgCl2
6 µl 1 mM EGTA
6 µl 1 mg/ml PKC ε peptide (Cat. No. 539562)
0.6 µl 10 mM ATP
6 µl lipid mixture
0.1 µl γ-ATP (5 mCi/ml)
32.9 µl distilled H2O
Total volume should be 60 µl. Prepare a sufficient reaction mixture for all assays to be performed.
Reaction:
1. Dispense 60 µl of reaction mixture into each tube and incubate at 30°C.
2. Dilute the PKC δ stock solution (500 ng/µl) to between 20-50 ng/µl in 10 mM Tris-HCl, pH 7.5, 5 mM DTT, and 0.01% TRITON®: X-100 detergent.
3. Add 2 µl of diluted enzyme to each tube.
4. Incubate for 10 min.
5. Stop the reaction by adding 6 µl of 5% phosphoric acid.
6. Incubate on ice for 5 min.
7. Spot 55 µl from each tube to phosphocellulose membranes and allow to dry.
8. Wash the membranes 3X with 5 ml of 0.5% phosphoric acid per filter.
9. Transfer membranes to a scintillation vial and count.
Note: This protocol is meant to serve as a guideline and may need to be modified for specific applications.
Purity
≥95% by SDS-PAGE
Specific activity
≥800 units/mg protein
Unit definition
One unit is defined as the amount of enzyme that will transfer 1 nmol of phosphate to PKCε Peptide Substrate (Cat. No. 539562) per min at 30°C, pH 7.5.
Storage
Avoid freeze/thaw ≤ -70°C
Do Not Freeze
Ok to freeze
Special Instructions
Following initial thaw, aliquot and freeze (-70°C).
Toxicity
Standard Handling
References
Hug, J. and Sarre, T.F. 1993. Biochem. J. 291, 329. Kazanietz, M.G., et al. 1993. Mol.Pharmacol. 44, 298. Koide, H., et al. 1992. Proc. Natl. Acad. Sci. USA 89, 1149.
