I have been preparing protein extracts from roots by grinding roots into PBS buffer, incubating on ice for 45mins, spinning out the pellet, then collecting the supernatant and resuspending the pellet. I have done the grinding at room temp in the eppendorf tubes and extracted very little protein (estimated visually from Ponceau-stained membrane). I also ground the root material into powder in liquid nitrogen before the extraction into PBS; I yielded more protein but still relatively little all together.

One issue I noticed is that no matter how I grind the roots, the never dissolve into a homogeneous (or nearly homogeneous) mixture like leaves.

Any suggestions for how to best grind the roots or any good extraction protocol?