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
will cuvette hold 250 or 500ul or more.
if 250 or 500ul, it should be fine. you can maintain the substrate concentrations accordingly and accordingly the enzyme or lysate if it is with lysates. and make up will assay buffer.
If the assay buffer is too much, you can dilute the lysate with the lysis buffer, if the components do not interfere with the assay conditions. But any ways you have to perform few trial experiments to make sure that the system is in place.
one technical aspect would be to raise the position of the cuvette from the base of the spec and make sure that the light source path still passess through the solution in the cuvuette.
by doing this you can reduce the volume to be used in the cuvette.
my cuvette has max vol of 3 ml, n for readings minimum vol required is 1.5 ml. raising the cuvette wont work as the cuvette is covered in the chamber and if its not placed it doesnt get closed properly.
yes i know i have to go by some trails, but the only problm is substrates r is too less in quantity n too costly... i cant afford to waste much in trails, so i want to be double sure of evrything before i start the assay. anyways thanks fr ur suggestion.
in that case, i do not think it's a good idea to try this out. you should try with a low volume cuvette, 0.5ml. i think it would be wise to invest in cuvette rather than reagents.
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