Diatom Chloroplast Isolation Steps
Grow diatom culture in autoclaved seawater media (e.g. f/2 media).
[Protocol was developed with cells grown in f/2 medium to a density of ~1x106 cells/mL - optimization may be necessary for cells achieving a lower cell density]Gently filter culture through a 2 μm 47 mM filter.
Periodically, as filtration slows due to growing biomass on the filter, place filters in 50 ml of media used for growth to gently was cells from filter.
Repeat until all culture is filtered and cells resuspended.
Remove filters from tubes and centrifuge @ 1200 x g for 5 minutes to pellet cells.
Gently decant media.
Resuspend pellet in ~7ml 10M NH4F. 
Vortex periodically while incubating tube on ice for 10 minutes.
Centrifuge @1200 x g @ 4°C for 5 min and pour off NH4F.
Resuspend and rinse pellet 3x with isolation buffer (5-7 mls), centrifuge @1200 x g @4°C in between rinses.
Resuspend pellet in 1 ml cold isolation buffer.
Sonicate @ 10W for 10 s. 
Prepare 2 two-step Percoll gradients (in 15 ml COREX tubes).
Add 2 ml Bottom Layer Solution (80% Percoll) to 15 ml corex tube.
Apply 10 μl of gel loading dye the Top Layer Solution (40% Percoll) and gently add 4 ml Top Layer to the 80% Percoll layer using a Pasteur Pipette.
Load homogenate from step 9 (Silica Frustrule Removal) carefully onto the top of each two-step Percoll gradient with a Pasteur pipette.
Centrifuge in swing-out rotor at 1500 x g for ten minutes brake off (@4°C).R
ecover intact chloroplasts (bottom layer) using a Pasteur Pipette.
Add to sterile 15 ml corex tube.
Wash twice with cold isolation buffer (without added BSA) as below:
Add buffer.
Centrifuge in a swing out rotor at 1000 x g @4°C for five minutes (brake on).
Discard Supernatant.
Use a soft paintbrush to carefully resuspend pelleted plastids in a small amount of cold isolation buffer (or alternate preservation buffer of your choice). 
[soft paintbrush is specified so that you do not disrupt the fragile exposed plastids]. 
Use phase contrast microscopy to visualize and confirm successful extraction of intact chloroplasts.
Plastid enrichment can be followed by subsequent protein extraction and gel electrophoresis on SDS PAGE gels for relative enrichment of RuBisCO in the plastid fraction.
