CASPASES and APOPTOSIS

Detecting Caspase Activity

How to Detect Activated Caspase on Western Blot?
*Band would be smaller (lighter) than original inactive caspase.
-Caspases are cut into 2 pieces 2 remaining caspases dimerize, held together by non-covalent bonds.
-SDS-PAGE disrupts noncovalent bonds Separates the 2 caspase dimers complex See lighter band
that represents each dimer half.

*Default: SDS-PAGE always starts off Western Blot.

-CANNOT detect whole multiprotein complexes.

Detecting Apoptosis
What is Being Detected in the Fluorescing Apoptotic Cells?
-CUT DNA ENDS.
-Its one of the major outcomes of Apoptosis.

NOT
-Phosphorylated Caspases
-Caspases are activated by cleavage.
-Kinases DO NOT target Caspases.
-Cleaved Nuclear Lamins
-Do get cleaved, but theres no fluorescent marker that
labels uncleaved vs. cleaved ends of a lamin.
-Cytosolic Cytochrome C Exists.
-Too small (in image) to its location.
*Image too zoomed out, shows whole cells.
-Phosphotidyl Serine in Outer Plasma Membrane leaflet
Nonexistent technique.
-Membrane too thin to determine which leaflet Phosphotidyl serine is on.

TUNEL Assay: Terminal Deoxynucleotidyl Transferase (TdT) dUTP Nick-End Labeling

*TdT (Terminal Deoxynucleotidyl Transferase) = Enzyme that adds deoxy-U to a cut DNA
end, or any DNA end.
1. Fix tissue / section.
2. Add TdT enzyme + Fluorescent UTP
-Fluorescent UTP = Adds to ends of DNA (cut or not) becomes fluorescent UMP.
3. If cell has done Apoptosis Lots of cut DNA Lots of cut DNA ends.
4. Fluorescent UTP adds to lots of cut DNA ends Visualized as lots of Fluorescent UMP.

Localization of Fluorescent Cytochrome C Assay

*Look at cells much closer, cell-to-cell evaluation.
-Purpose: Look at localization of Cytochrome C.
 Observations
-Control Cells: Cytochrome C labeling (green) is punctated over cytoplasm.
-Mitochondrial labeling (red) overlaps with Cytochrome C.
-UV-Treated Cells: UV applied Damages DNA & cells Apoptosis.
-Cytochrome C (green) is spread throughout cytoplasm Shows Apoptosis.