Hello.
I need to detect a protein that is present only in larvae (and compare them to adults). The problem is that I want to load equal amounts of protein. And I want to make Bradford assay. I tried to lyse worms with ripa buffer but that didn't work. It didn't lyse even bacteria. (I boiled samples for 10 min.) The amount of worms I get is too small for sonication.
What approaches can I use without sonication?
Dear Maria,
You have to break the skin barrier. Try grinding it in a glass grinder (looks a bit like the old mortar in an apothecary from 200 years ago) and then redissolve in LSB directly. It is not compatible with Bradford assay but the gel may tell you if you have your protein (Coommassi, WB, etc).
Worms are tough and breaking their skin is impossible with a RIPA buffer. I doubt boiling them with LSB will do the trick but you may try.
Best regards,
Wes
Dear Maria,
You may try, dilute the volume with lysis buffer and adjust the volume which will be compatible for sonication. and use the PIC inhibitor during lysis (sonication).
Best regards,
Abodh
I used to do protein extractions with C. elegans a lot. We usually used a ball mill / vibrating mill (https://www.retsch.de/images/42c82b43b352ab3eb4e0bbd74e3148cf/x300/max/alpha.webp), which usually worked quite good
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