1. Antibody to Single Stranded DNA
A widely used cytochemical technique for evaluation of DNA damage associated with apoptosis is the terminal deoxynucleotidyl transferase-mediated in situ end labeling or TUNEL assay. However the TUNEL assay has its drawbacks in that false positive staining makes the assay unreliable as a marker for apoptosis 1-5. A more universal and specific marker for detecting apoptosis associated DNA damage is to measure the morphological changes in nuclei that reflect chromatin condensation into compact masses. 6-7. Further biochemical and cytochemical studies have demonstrated the increased susceptibility of apoptotic DNA to thermal denaturation. Analysis of nuclei by scanning calorimetry to detect thermal induced DNA denaturation and analysis of DNA fragmentation by electrophoresis have shown that intact apoptotic DNA is susceptible to denaturation at lower temperatures then that of non-apoptotic cells 8.
2. Antibody to Active Caspase 3
Apoptosis is an evolutionarily conserved form of cell suicide, which follows a specialized cellular process. The central component of this process is a cascade of proteolytic enzymes called caspases. These enzymes participate in a series of reactions that are triggered in response to pro-apoptotic signals and result in cleavage of protein substrates, causing the disassembly of the cell 13. Caspases have been identified in organisms ranging from C. elegans to humans. The mammalian caspases play distinct roles in apoptosis and inflammation. In apoptosis, caspases are responsible for proteolytic cleavages that lead to cell disassembly (effector caspases), and are involved in upstream regulatory events (initiator caspases). An active caspase consists of two large (~20 kD) and two small (~10 kD) subunits to form two heterodimers which associate in a tetramer 14-16. As is common with other proteases, caspases are synthesized as precursors that undergo proteolytic maturation, either autocatalytically or in a cascade by enzymes with similar specificity 17. Caspase 3, also known as CPP-32, Apopain or Yama, is a key effector caspase in the apoptotic pathway 18. It is present in many different cell lineages and is responsible for the cleavage of a variety of molecules such as poly ADP-ribose polymerase (PARP), protein kinase Cd, actin and DNA-dependent protein kinase 19-20.
- Much more Reliable as compared to TUNEL assays – No False positive signals.
- Multiple parameter detection of DNA damage and caspase 3 activity simultaneously.
- Readout – Flow cytometry, Fluorescence 96 well plate reader, Fluorescence microscope.
- Yields both quantitative and qualitative results. Gives a strong positive signal.
- Ease Of Use: Dual parameter results to confirm apoptosis in cells.
- Compatible with human, mouse, rat, bovine, porcine species.