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Proteasome assay


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6 replies to this topic

#1 poonam thakur

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Posted 13 July 2010 - 05:36 AM

Hi
I wish to do the proteasome assay using the flouregenic substrates Suc-LLVY-AMC, Z-LLE-AMC, Boc-Leu-Arg-Arg-AMC. All the protocols i have read are using well plates. But my spectroflourometer does not have a plate reader attachment. i want to upscale the assay to the cuvette. will diluting the same amounts as required in wel plates with the assay buffer give me the reading??? also what are the other precautions i must keep in mind while working up.. If anybody can provide me with a detailed protocol it would be really really helpful

Edited by poonam thakur, 13 July 2010 - 05:40 AM.


#2 mdfenko

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Posted 13 July 2010 - 06:54 AM

no. you must scale up rather than dilute (you may get a reading but it will be a lot less intense than that from the wells).

or get access to a fluorescence plate reader.
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#3 poonam thakur

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Posted 13 July 2010 - 05:47 PM

hi thanks... have u done this assay or a similar one before??

#4 mdfenko

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Posted 14 July 2010 - 06:33 AM

i've done a lot of different protease assays, including some proteosome work.

a friend in my former lab had done more work with proteosomes. we did individual assays rather than plate based and made our own solutions.

here is the reference for a paper that we published. it will give assay conditions:

Malik, M.N., W.D. Spivack, A.M. Sheikh and M.D. Fenko, “The 26S Proteosome in Garlic (Allium Sativum): Purification and Partial Characterization”, J. Agric. Food Chem., 52, 3350-3355 (2004)

here is a pdf of the galley proof:

Attached Files


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#5 poonam thakur

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Posted 14 July 2010 - 05:14 PM

:(
hi thanks a lot... But one thing i am wondering about is have you plotted the calibration curve using AMC?? Coz normally people express activity as nM of AMC released/mg protein/min... or have you taken certain amount of AMC released as the activity units?
Since you have done protease assays i think you can answer one more question for me... We will be doing proteasome assay in the crude tissue lysates without any further purification of proteasome.. the other proteases present in the lysate might also act to cleave the substrate hence not showing the pure proteasome activity.. Will adding protease inhibitor cocktail to the mixture help??
Also is there any way i can distinguish between 20S and 26S proteasome activity?

#6 mdfenko

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Posted 16 July 2010 - 11:23 AM

for our purposes, we were just using raw fluorescence minus a blank to localize the proteosome. if we wanted to determine the moles of substrate utilized we would have run an amc standard curve along with the assay.

the substrate you use should be reasonably specific for proteosomes and be virtually unaffected by other proteases. if you do choose to use an inhibitor cocktail, then you will have to ensure that none of the inhibitors will affect the proteosome.

as told in the paper, the effect of the presence of sds in the assay compared to one without sds will let you know if you have 20s or 26s.
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#7 poonam thakur

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Posted 23 July 2010 - 10:35 PM

Thanks a lot for the tips




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