Native gels for proteasome activity assay - (Mar/28/2008 )
In my lab we are recently trying to measure proteasome activity assay in native gels. We use cell extracts (hippocampal neurosn, DIV 7 and 15); for the condition of the gel we are using 3.5% for the resolving and 2.5% for the stacking gel. Furthermore, we have purified proteasome as positive control (Biomol). After the electrophorese we incubated the gel with a chemotrypsin-fluorescent substrate during 45min. Unfortunatly, we don't have a strong signal ( no signal in our samples), and a weak signal from the purified proteasome.
Do you have some clue, or even a bettre protocol.. we are really open to new ideas.
thanks in advance
i recommend a gradient gel to sharpen (concentrate) your bands. 3-5% should do, but will be hard to handle (not as hard to handle as 3.5%).
what buffer formulation are you using?
check the fora for other formulations.
if the control showed then yours should have but, are they the same size?
how pure is yours?
these factors can effect your detection.
Hey!!Thanks a lot for the tips!!
Actually i don't have that much problem handling a 3.5% gel..
About my buffer, i'm using TBE (Tris 90mM, acid boric 80mM and EDTA 0.1mM + 1mMDTT, pH 8.3). This is based in the book: Methods in molecular bioloy.
The strange is that if the positive control does appear, why my sample are not?! I am expecting proteasome in both have the same size. I don't know if the fcat that i'm working with whole protein cell extract is afecting the migration in the gel.. i mean... does my sample need more time?!
I've looked in this forum, and i guess that gradient gels are a good option. I'll discuss that with my boss.
Any way, does anyone as a protocol for native gels?!
Thanks in advance
Oh!! about the marker, i couldn´t find any.. i want one that is prestained, so i can look for the migration of the samples...and proteasome is about 800 kda... not easy to find that
there are protocols for native gels posted in these fora. just search.
we use a tris-glycine buffer system for native page.
prestained markers are for denaturing page (sds) and are in the sample buffer for that. they can't be used for native page.