The 20S Proteasome Activity Assay Kit from Millipore provides a simple and reproducible method to measure the activity of the proteasome in cell lysates or purified enzyme preparations. The activity of the proteasome is quantified by measuring the amount of fluorescence produced by cleavage of the fluorogenic substrate LLVY-AMC. The peptide LLVY, a substrate of the chymotrypsin activity of the 20S proteasome, is bound to the fluorophore 7-Amino-4-Methylcoumarin (AMC), which when released by proteasomal cleavage, emits fluorescent light. This fluorescence can be read using a 380 nm excitation and a 460 nm emission filter in a fluorometer.
The kit contains all of the reagents needed for the assay except for DMSO and 96-well plates. A 37oC incubator and a fluorometer equipped with a 380/460 nm filter set are needed to develop and read the results of the assay. The kit contains the assay buffer, the proteasome substrate, a 20S proteasome positive control and AMC standard needed for the quantification of results. The kit also provides the proteasome inhibitor Lactacystin, which can be used for screening of samples. Most components of the kit can be stored at -20oC once reconstituted, and the solutions can be stored for 3-6 months. One kit provides enough reagents to perform 100 assays.
The assay procedure is very simple and straightforward, and explained clearly in the kit’s accompanying instructions. The assay buffer, sample and proteasome substrate are mixed together, preferably in a 96-well fluorometer plate. The mixture is incubated for 1-2 hours at 37oC to 60oC, after which the fluorescence is read in a fluorometer. (The reason for this broad range in incubation temperature is not explained in the instructions, but it could be intended to give flexibility in the incubation times. I used a 37oC incubation which seemed to give very good results.) A fluorogenic standard curve can easily be generated using the provided AMC standards diluted in assay buffer, which allows for the accurate calculation and expression of results. A 20S proteasome activity standard curve can also be generated by making a dilution series with the provided proteasome positive control.
I used Millipore’s 20S Proteasome Activity Assay Kit to detect differences in the basal proteasome activity levels of a panel of 7 different breast cancer cell lines. When treated with proteasome inhibitors, different breast cancer cell lines show a range of IC50 values in cell viability tests. I attempted to establish a correlation between basal proteasome activity and resistance or sensitivity to proteasome inhibitors. The 20S Proteasome Activity Assay from Millipore helped me show that not only do cancer cell lines show a range of basal proteasome activities, but also that a positive correlation exists between proteasome activity levels in untreated cells and resistance to proteasome inhibitor treatment.
I found this particular kit very easy to use and the results obtained very reproducible. Each assay was performed in 8 replicates and each cell line and inhibitor dose was tested a minimum of three times, with very consistent results and small standard error bars. The preparation of the assay mix is very easy once all the calculations are made. The assay works at its optimum when 50 ug of whole cell lysate is used per assay, which requires prior determination of protein concentration in the lysates. The assay instructions do not include guidelines for optimum protein concentration or preparation of cell lysates. I extracted protein from cells using a buffer containing 1% NP40, 1% deoxycholate, 100 mM NaCl at pH 7.5, and diluted samples to their final concentration using the provided assay buffer. The composition of the assay buffer is included in the instructions and is therefore easy to reproduce. Reading the results is very straightforward after a given incubation time, which needs to be optimized for each sample and protein concentration used, since samples continue to develop fluorescence over time. The analysis of results is easy using an AMC standard curve generated with the same conditions as the ones used for the samples to be assayed. The kit’s only disadvantage is possibly the cost, but the reproducibility and ease of use make it a good investment.