TITLE: Protein Extraction of Symbiodiniaceae freshly isolated from Anthopleura elegantissima
AUTHORS: Virginia Weis

[DESCRIPTION]
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[STEPS]
?. [Isolation of algal pellet from whole animal]
Obtain 25 frozen small Anthopleura elegantissima (You could use 10 medium or 3 large animals also). Adjust the number of animals as needed.
?. [Isolation of algal pellet from whole animal]
Partially defrost and cut off pedal discs with a razor blade (keep the algal-rich tentacle crown).
?. [Isolation of algal pellet from whole animal]
Grind animals in mini-food processor or glass-teflon grinder in 30 ml FSW.
?. [Isolation of algal pellet from whole animal]
Divide into 4-50 ml tubes, rinse processor and include rinsate.
?. [Isolation of algal pellet from whole animal]
Spin for 6 min at 2500 xg at 4˚C.
?. [Isolation of algal pellet from whole animal]
Rinse and re-spin approximately 5 times.  Vigorously resuspend pellet via vortex each time.
?. [Isolation of algal pellet from whole animal]
Filter each tube of algae through 2 layers of cheesecloth to remove large chunks of tissue
?. [Isolation of algal pellet from whole animal]
This procedure should yield about 6 ml of algal pellet for A. elegantissima.
?. [Extraction of algal protein]
Work with 1.5 ml of above algal pellet.  Freeze the remainder.
?. [Extraction of algal protein]
To this 1.5 ml, add 10 ml of FSW with 2% triton X100.  Resuspend algae via vortex.
?. [Extraction of algal protein]
Spin at 2,500 xg for 6 min at 4˚C.  Supernatant should have greenish-yellow tint.  Remove and discard supernatant.
?. [Extraction of algal protein]
Rinse pellet once with 10 ml of FSW with 2% triton X100 and spin again at 2,500 xg for 6 min at 4˚C.
?. [Extraction of algal protein]
Remove and discard supernatant and add 3.75 ml of extraction buffer (with PIC). Resuspend algae and place suspension in a glass culture tube.
?. [Extraction of algal protein]
Add 1-2 ml of glass beads (acid washed).
?. [Extraction of algal protein]
Vortex suspension for 30 sec and then place on ice for 30 sec.
?. [Extraction of algal protein]
Repeat vortex and icing a total of 20 times
?. [Extraction of algal protein]
Remove suspension, away from glass beads and place in microfuge tubes.
?. [Extraction of algal protein]
Spin at 15,000 rpm in microfuge for 5 min at 4˚C.  Resulting supernatant should be a deep, clear orange.
?. [Extraction of algal protein]
Determine protein concentration with Bradford or other protein quantification assay.
?. [Mix extraction buffer]
Recipe (adjust volumes as needed):50 mL - 100 mM Tris50 mL - 10 mM EDTA50 mL - 100 mM NaClAdjust pH of the buffer to 7.4.The day of use, add 1 aliquot of Protease Inhibitor Cocktail (PIC) to 10 mL of buffer.A few options for PIC: 1. https://www.sigmaaldrich.com/catalog/product/sigma/p9599?lang=en®ion=USP9599 from Sigma-Aldrich (extraction from plant tissue)2. https://www.sigmaaldrich.com/catalog/product/sigma/s8820?lang=en®ion=USS8820 from Sigma-Aldrich (general use)
?. [Gather materials   ]
1. 2% Triton X100  in FSW2. FSW3. Acid washed glass beads (400-600µm in diameter): https://www.sigmaaldrich.com/catalog/product/sigma/g8772?lang=en®ion=US        G8772 from Sigma-Aldrich
?. If you are working with an algae culture, skip to step 12.