I am working on entomopathogenic nematodes. What are the best method to extract the total lipid carbohydrate and proteins from these tiny nematode? Is there any kit available in the market for there extraction? Thanks in advance.
For total worm protein for an SDS PAGE gel, here is my protocol, in case this helps you:
1.) Thaw a 50-200ul worm pellet in a microfuge tube from the -80 and add 1:1 volume of NSB (nematode solubilization buffer): 0.3% ethanolamine, 2mM EDTA pH8, 1mM PMSF, 5mM DTT, 2X protease inhibitor cocktail.
2.) Use a long glass Pasteur pipet to transfer to a 3ml glass Potter-Elvehjem homogenizer, and homogenize with a rotor for 20 slow strokes at 400 RPM.
3) Pipet back into the microfuge tube and place in 80 degree heat block for 10min.
4) Add 1:1 volume of 2X SDS buffer: 100mM Tris HCl pH 6.8, 20% glycerol, 4% SDS. Heat again for 10 min and vortex hard.
5) Spin at max speed for 6 min. Remove the cleared lysate sup to a clean tube.
6) Use an assay such as BioRad DC Assay to quantify proteins (while samples are kept on ice).
7) Add BME to a final concentration of 5% and bromophenol blue to a final concentration of 0.1% and vortex before loading on a gel.
For nematode lipids, you may want to consult Carissa Olsen, whose lab is currently using improved isolation protocols .
http://www.sciencedirect.com/science/article/pii/S1550413108002489
for extraction methods of total lipid you should use the n-hexan extrtction by Soxhlet ..
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