S-100 HeLa Degradation Kit

(50 μl reaction)

1. Thaw all enzyme/protein solutions on ice. Inhibitor control in DMSO must be thawed at room temperature. Briefly vortex all solutions prior to use.

2. Add 26 μL of HeLa S-100 (200 μg, 4mg/ml final)

3. Add 1.25 μL MG-132 (5 μM final) for NEGATIVE CONTROL ONLY! DO NOT ADD INHIBITOR TO DEGRADATION REACTION. Vortex and allow

inhibitor and HeLa S-100 to pre-incubate for 15 minutes at room temp.

4. Add 5 μL of ubiquitin solution (600 μM final)

5. Add substrate at desired concentration

6. Bring reaction volume to 45 μL with desired buffer. (50 mM Hepes, pH 7.6 recommended)

7. Initiate conjugation/degradation reaction with the addition of 5 μL of ERS

8. Vortex and incubate at 37oC. An initial time course is recommended to determine optimal activity and signal. Degradation is typically seen within 3-4 hours.

9. Depending on detection method, take appropriate quantity of the reaction at desired time and compare to proteasome inhibited (PI) reaction. If degradation is not inhibited by the PI, the detected degradation is most likely NOT due proteosomal degradation of the substrate.

Assay Considerations:

The S100 fraction contains all the necessary components to conjugate ubiquitin to substrate proteins, and also the components necessary for the degradation of these substrates by the proteasome. These enzymes/proteins include ubiquitin, E1, E2s, E3s (ligases), isopeptidases (UCHs) and proteasome (26S). Protein substrates of choice can

be added exogenously or if the protein of interest is already contained in the S100 fraction, its degradation may be tracked via immuno-detection.

Tracking degradation of the target protein depends on the detection method and user preference. Below are some examples of routine methods:

• Visualization of signal band on SDS-PAGE and subsequent disappearance

that is blocked by the control proteasome inhibitor. Detection of the substrate

band can be via radio-label or immuno-detection.

• Quantitative readout on the TCA soluble peptides produced over time that is blocked by the control proteasome inhibitor. This method is exclusively for

radio-labeled substrates.

If the approach is to add a substrate protein and track the decrease of a SDSPAGE band, care must be taken to add substrate to an amount that gives a clearly defined signal without the masking of the disappearance of the protein band which signifies degradation. Typical degradation rates will result in catabolism of 5-20% of the added substrate protein. If too much substrate is added, the gel signal will potentially hide the degradation of the 20% mentioned above. The ultimate optimal amount of substrate to be

added must be determined empirically. If the substrate protein is already contained in the S100 and is being assayed by immuno-detection, these considerations do not apply. The following assay protocol is for the set-up and execution of the experimental proteasome degradation. Final concentrations of substrate and time courses must be determined experimentally for each individual protein. Detection methods must also be

optimized based on activity, substrate quantity and time. The removal of ERS or the addition of a proteasome inhibitor is viable negative controls for demonstration of a UPP mediated event. Quantities of supplied reagents or reaction volumes may be changed to suit individual needs.