Analysis Note

Partially purified recombinant caspase 3 with a full length HIS-6-tag. Approximately 10-20% caspase 3 protein. The remainder is E.Coli proteins. Approximately 0.8-2 mg total protein/U of activity; E.Coli proteins have no reactivity.

Application

Western blot: 1-5 µL of stock per lane. Protein is full length caspase 3 with His-Tag. It is autoactivated thus active caspase subunits will be seen

and 17kDa + 12kDa (large and small subunits) can be seen depending upon the antibody. Small subunit is extremely difficult to detect in most westerns. The size of inactive enzime is ~32 kDa.

Activation: Not needed. When expressed in E. coli, caspase-3 spontaneously undergoes autoprocessing to yield the subunits characteristic of the active enzyme.

Disclaimer

Unless otherwise stated in our catalog or other company documentation accompanying the product(s), our products are intended for research use only and are not to be used for any other purpose, which includes but is not limited to, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses or any type of consumption or application to humans or animals.

General description

Specificity: Caspase-3 (also known as CPP32, Yama and apopain) is a member of the interleukin-1beta converting enzyme (ICE) family of cysteine proteases. Caspase-3 exists in cells as an inactive 32 kDa proenzyme. During apoptosis procaspase-3 is processed at aspartate residues by self-proteolysis and/or cleavage by upstream caspases, such as caspase-6 (Mch2), caspase-8 (FLICE) granzyme B. The processed form of caspase-3 consists of large (17 kDa) and small (12 kDa) subunits which associate to form the active enzyme. The active caspase-3 has been shown involving in the proteolysis of several important molecules, such as poly (ADP-ribose) polymerase (PARP), the sterol regulatory element binding proteins (SREBPs), focal adhesion kinase (FAK), and others.

The partially purified* active recombinant human caspase-3 protein with full-length His-6 tag was expressed in E. coli. The expressed Caspase-3 spontaneously undergoes autoprocessing to yield the subunits characteristic of the native enzyme. The recombinant caspase-3 preferentially cleaves caspase-3 substrates (e.g., DEVD-AFC or DEVD-pNA). In combination with caspase-3 activity assays, the active recombinant caspase-3 is very useful in the biological screening of caspase inhibitors. The recombinant enzyme can also be used as a positive control in caspase assays or in determining the specificity of caspase substrates.

*10-25% caspase-3 protein by gel analysis, also contains E. Coli proteins. Approximately, 0.8-2 µg total protein / unit of activity. GenBank # U13737.

Legal Information

CHEMICON is a registered trademark of Merck KGaA, Darmstadt, Germany

Physical form

Lyophilized. Reconstitute to 1 unit/µL with PBS containing 15% glycerol.

Partially purified recombinant caspase 3 with a full length HIS-6-tag. Approximately 10-20% caspase 3 protein. The remainder is E.Coli proteins. Approximately 0.8-2 mg total protein/U of activity; E.Coli proteins have no reactivity.

Storage and Stability

Maintain unopened and rehydrated product at -70°C in undiluted, freeze-thaw protected, tightly sealed aliquots. Avoid repeated freeze/thaw cycles. Product stable for 6 months from date of purchased if handled appropriately.

Unit Definition

Specific Activity: One unit of the recombinant caspase-3 is the enzyme activity that cleaves 1 nmol of the caspase substrate DEVD-pNA (pNA: pnitroanaline) per hour at 37°C at saturated substrate concentrations.