| Caspase-3 is one of the key executioners of apoptosis, being responsible either partially or totally for the proteolytic cleavage of many key proteins such as the nuclear enzyme poly (ADPribose) polymerase (PARP) (1). Activation of caspase-3 requires proteolytic processing of its inactive zymogen into activated p17 and p12 subunits. Cleavage of caspase-3 requires aspartic acid at the P1 position (2). |
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| Catalog # | C2087-20J |
| Kit Description | Apoptosis Marker: Cleaved Caspase-3 (Asp175) IHC Detection Kit is a “ready to use” system designed to detect the activation of caspase- 3 in human tissue and cell preps by immunohistochemistry. The kit utilizes the ABC immunoperoxidase method to detect endogenous levels of caspase-3 protein. Prediluted Cleaved Caspase-3 (Asp175) Antibody is bound by a biotinylated secondary antibody. Avidin DH and biotinylated horseradish peroxidase are complexed by mixing defined amounts prior to use, and the mixture subsequently binds the secondary antibody. The macromolecular complex is localized by incubation with NovaRed enzyme substrate. The prediluted primary antibody, along with the ABC system, allows the user to examine caspase-3 localization consistently and offers the highest sensitivity with the lowest background. |
| Kit Specificity/Sensitivity | Apoptosis Marker: Cleaved Caspase-3 (Asp175) IHC Detection Kit detects endogenous levels of the large fragment (17/19kD) of activated caspase-3 resulting from cleavage adjacent to Asp175. The cleaved caspase-3 antibody in the kit does not recognize full length caspase-3 or other cleaved caspases. The antibody was produced by immunizing animals with a synthetic peptide corresponding to amino-terminal residues surrounding Asp-175 of human Caspase-3 and was purified by Protein A and peptide affinity chromatography. |
| Intended Use | This kit was developed for and is recommended for immunohistochemistry only. One kit is good for use with 150 slides. |
| Species Cross-reactivity | Human and mouse. Expected to react with rat based on 100% sequence homology. Species cross-reactivity has been determined by Western Blot. |
| Kit Components | C2087-20J1: Peroxidase Quench, 1x19.5ml |
| C2087-20J2: Blocking Solution, 1x15ml |
| C2087-20J3: Cleaved Caspase-3 Antibody, Pre-diluted, 1x15ml |
| C2087-20J4: Negative Control, Pre-diluted, 1x15ml |
| C2087-20J5: Secondary Antibody (Biotin), 1x15ml |
| C2087-20J6: Reagent A, 1x850ul |
| C2087-20J7: Reagent B, 1x1850ul |
| C2087-20J8A: NovaRed™ Substrate A, 1x600ul |
| C2087-20J8B: NovaRed™ Substrate B, 1x600ul |
| C2087-20J8C: NovaRed™ Substrate C, 1x600ul |
| C2087-20J8D: NovaRed™ Substrate D, 1x900ul |
| C2087-20J9: Cleaved Caspase-3 Blocking Peptide, x100ul |
| Notes: 1. The kit is also supplied with a mixing bottle. 2. NovaRed™ is a trademark of Vector Laboratories |
| Reagents Required But Not Supplied | Xylene |
| Ethanol, 100% and 95% |
| Distilled Water |
| Hematoxylin (optional) |
| Mounting Medium |
| Posphate-Buffered Saline + 0.1% Tween-20 (PBS/T). To prepare 1 liter: Add 8g sodium chloride (NaCl), 0.2g potassium chloride (KCl), 1.44g sodium phosphate, dibasic (Na2HPO4) and 0.24g potassium phosphate, monobasic (KH2PO4) to 800ml dH2O. Adjust pH to 7.4 then bring volume to 1 liter. Add 1ml Tween-20. Mix well. |
| 0.01M Sodium Citrate Buffer, pH 6.0. To prepare 1 liter: Add 2.94g sodium citrate trisodium salt dihydrate (C6H5Na3O7•2H2O) to 800ml dH2O. Adjust pH to 6.0, then bring volume to 1 liter. |
| Storage and Stability | Store kit at 4°C. Components are ready to use and should not be aliquoted. |
| Important Note | This product as supplied is intended for research use only, not for use in human, therapeutic or diagnostic applications without the expressed written authorization of United States Biological. |
