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 .
