Experimental pitfalls

Experimental variablesPitfall
 ATP and magnesiumStability of the 26S complex is dependent on a high ATP/ADP ratio. For monitoring 26S activity, an adequate amount of ATP and MgCl2 in a 1:5 ratio (we typically use 2 and 10 mM, respectively) should be present in all buffers to maintain the stability of the 26S proteasome complex. At the same time, the levels of ADP should be kept at a minimum
 GlycerolGlycerol in the buffer stabilizes and maintains the closed, latent gate. Typically, purification buffers for proteasomes contain 10% glycerol, and assay buffers contain 5%
 SDSAddition of 0.02% SDS to assay buffer mildly stimulates opening of the latent 20S gate
Proteasome populationProteasomes are present as individual 20S particles and 26S particles (singly caped 20S-19S and doubly capped 19S-20S-19S). The 26S complex can dissociate into the 19S and 20S constituents over time, after a freeze/thaw, or in response to buffer condition changes
When performing experiments with purified 26S proteasomes, it is important to determine the ratio of intact 26S and free 20S proteasomes. This is commonly accomplished by native PAGE electrophoresis as described in the text
MicroplateThe type of microplate (e.g., untreated vs. nonbinding surface treatments, polypropylene vs. polystyrene) can affect the observed activity. Some substrates (e.g., GFP) interact with and “stick” to untreated plate surfaces. Some compounds or small substrates may bind to the plate surface and reduce the effective concentration in the assay. It is worthwhile to confirm results with new compounds or substrates by using two or more types of plates. Bovine serum albumin can be included in the buffer to prevent nonspecific binding to the plate
Proteasome gating Experiments probing the role/regulation of the proteasome gate might be facilitated by using the “open-gate” 20S mutant, α3ΔN
Suc-LLVY-amc has been shown to mildly stimulate gate opening, so another substrate (e.g., Ac-nLPnLD-amc) should be used in conjunction