Hi Yutthana,

For crude total protein extraction from C.reinhardtii (intact cells),

I use the following Extraction Buffer (EB) (ingredients in bold are to be added fresh just before use):

• 100 mM HEPES, pH 7.5
• 150 mM NaCl
• 4 mM EDTA
• 10 % v/v Glycerol
• 0.4 mM PMSF
• 0.1 v/v % Triton - X - 100
• 1 mM DTT ( can go up to 5 mM if needed )
• 1/2 tablet Protease Inhibitors Coctail (Complete mini, Roche)

I follow the procedure below:

• Grind in N2(l) until fine powder.
• add 400 μl of EB in approx 85 mg of tissue powder
• Incubate samples @ 4oC for 5 min with gentle agitation/rotation
• Centrifuge @ full speed (bench-top centrifuge) for 5 min @ 4oC (Use cooling centrifuge or place a centrifuge in the cold room)
• Transfer supernatant in a new tube (optional : centrifuge again to pelet any carry-over debris)
• Proceed to protein quantification and downstream handling

The above protocol works fine for me and is OK for Western Blots and IP . There may be considerable amounts of nucleic acids in the extract, but this has never affected my downstream experiments. For separating flagella... I have no experience. I suppose you will need to separate flagella from the cells in advance..

Best of Luck with your experiments,

Chris