Measurement of ROS in C elegans

Hello everyone,
I am performing ROS measurement nowadays and since different papers give different assays I am a bit confused.
I started taking 50 whole worms and 50 micromolar DCFDA, 1 hr incubation at 20 C. But since our lab doesnt have Fluorescence plate reader, I am taking readings on spectrofluorimeter in 1 ml cuvette, this method causes unequal no. of worms as during pipetting worms stick to the pipette tip, giving unexpexted results.
Then I went for lysis method where I am lysing worms in lysis buffer, and we have to report readings a fluorescence/ mg protein. Here the readings are coming fine but when I am doing Bradford assay, I cannot see any change in readings, despite unequal no. of worms and found that my sample buffer itself is giving so much of color.
So anyone can suggest any method or lysis buffer which I can use for isolating proteins in C elegans, that does not interfere with Bradford assay and also do not interfere with dcfda assay???


using triton x-100 0.01-0.1% or Tween in solutions or as a wash prevents the worms sticking to the pipette tips (see numerous worm methods in wormbook).

Without knowing what’s in your lysis buffer it’s difficult to say what it is that’s interacting with the Bradford reagents…

However, for a lysis buffer suitable for proteins you may find this link to an earlier discussion useful,


Not sure how your ROS measurement works with lysates, nor do I know what’s in your lysis buffer. But if it’s similar to what’s in whole cell lysis buffer for westerns, here’s what I can tell you about how that buffer effects Bradford assays.
Standard lysis buffers have dyes in it that you don’t need, make sure your lysis buffer has no dye in it (the purpose of the dye is simply to serve as a marker for you to see when a western blot is running). You don’t need it in your lysis and it interferes with Bradford.
SDS is the only remaining chemical in most whole cell lysate buffer that interferes with the Bradford assay, but you can still make it work. Yes you’re normalization curve will be compressed as the reaction is somewhat inhibited by the SDS, but if you normalize the amount of SDS in your protein standards to match the amount in your lysate you can get very accurate reading of protein concentration (do everything in 3x). Then it works fine every time.

Without knowing more about what you’re doing, I can’t assist more. Anyway, I hope this helps and good luck! :slight_smile:

The composition of my lysis buffer is 25 mM Tris HCl, pH 6.8, 10% glycerol, 10% b-mercaptoethanol, 4% SDS and 9 mM EDTA. Even I guess its because of SDS that Bradford assay is not working.
I tried the lysis buffer proposed by mcewan in the previous worm forum discussion but couldnt get sufficient protein. Its only after heating at 99 C for half hour I am getting protein, else I have tried liquid nitrogen, sonication, as well as mechanical disruption using syringe but no results.
Can anyone tell me in detail the protocol that might be working for you???
This will help a lot…
Thanks for the suggestions…:slight_smile:

forget bradford. use the bca assay to determine the protein concentration. bca does not react with sds. but exclude beta-mercaptoethanol from your lysisbuffer. then it should work fine.
cheers, oldcoffee