I am trying to measure 26s proteasome activities, but I cannot find a protocol in detail. I have two fluorogenic substrates, but I do not know how much volume to add. I found an article describing how to do this experiment, but I cannot get it online. It is called Monitoring activity and inhibition of 26s proteasomes with fluorogenic peptide substrates . It is published on Methods Enzymology. Can anyone can help me?
I used the protocol from promega, the proteasome glo
https://www.google.de/url?sa=t&rct=j&q=&esrc=s&source=web&cd=3&cad=rja&ved=0CEEQFjAC&url=http%3A%2F%2Fwww.promega.com%2Fproducts%2Fdrug-discovery%2Fprotease-assays%2Fproteasome_glo-assays%2F&ei=pjQtUejRKeSF4gSe74GQBw&usg=AFQjCNGWiYXdgK4hTUr5DDlz1nJwI6-C0g&sig2=2Li-hUtF9MculZTJK4Tbyw
The most difficult is to establish a good inhibition of the proteasome before performing measurement!
Let me know how it is working!
I followed the method mentioned in this paper-Increased proteasome activity in human embryonic stem cells is regulated by PSMD11. It is published on nature(doi:10.1038) . But it is too unclear , I try measuring the activities of 26S proteasome today , maybe a bad result, I can't understand it.
This is their protocol:
The in vitro assay of 26S proteasome activities was performed as previously described10.
-> well it is not the original paper we need
Cells were collected in proteasome activity assay buffer (50 mM Tris-HCl, pH 7.5, 250 mM sucrose, 5 mM MgCl2, 0.5 mM EDTA, 2 mM ATP and 1 mM dithiothreitol)
-> relatively clear
lysed by passing ten times through a 27-gauge needle attached to a 1-ml syringe. Lysate was centrifuged at 10,000g for 10 min at 4 °C.
-> ok
Approximately 15–25 μg of total protein of cell lysates were transferred to a 96-well microtiter plate (BD Falcon), and the fluorogenic substrate was then added to lysates.
-> they are using a kit from enzo, do you have that one BML-AK740-0001
To measure the chymotrypsin-like activity of the proteasome we used either Z-Gly-Gly-Leu-AMC (Enzo) or Suc-Leu-Leu-Val-Tyr-AMC (Enzo). We used Z-Leu-Leu-Glu-AMC (Enzo) to measure the caspase-like activity of the proteasome, and Ac-Arg-Leu-Arg-AMC for the proteasome trypsin-like activity. Fluorescence (380 nm excitation, 460 nm emission) was monitored on a microplate fluorometer (Infinite M1000, Tecan) every 5 min for 1 h at 37 °C.
-> follow instruction of the kit
http://www.enzolifesciences.com/BML-AK740/proteasome-20s-assay-kit/
Best
We have a protocol we're using for the development and testing of proteasome activity assay kits and novel substrates and inhibitors. It covers all three proteasome activities (chymotrypsin-like, trypsin-like and caspase-like) and utilises calibration standards that take into account the auto-hydrolysis of substrates such as Suc-LLVY-AMC during the course of a 30-60 minute assay at 37ºC.
Would be more than happy to provide a pdf copy if of interest?
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