Protein extraction from diatoms

Diatom Protein Extraction and Assay

Diatom culture samples were harvested by vacuum filtration onto glass fiber filters with 0.4 μm effective pore size.

Total protein was extracted using an MPBio FastPrep®-24 Instrument with the 24x2 rotor. Comparable results have been achieved using extraction instruments from PreCellys and SPEX.

Frozen filters were transferred to 2mL bead tubes containing MPBio lysing matrix D (SKU 116913050) and 700μL of 1X extraction buffer.

The extraction buffer was prepared from 4X protein solubilization buffer and 50X Pefabloc on the day of the extraction.

The 4X PSB contained 0.55M TRIS buffer, 0.3M LDS, 4.3M glycerol and 2mM EDTA.

The FastPrep was set to run at 6.5m/s for three one-minute cycles with the specified 24x2 rotor. The samples were held on ice for one minute between each cycle.

Following lysis, the samples were centrifuged for 3 minutes at 10 000 xg and the supernatant was assayed for total protein concentration using the BioRad DC protein assay kit with BGG known standards.

Courtesy of Jennifer Jeans, Mount Allison University.

Reference

Wu et al. (2011). Distinctive photosystem II photoinactivation and protein dynamics in marine diatoms. Plant Physiol. 156(4):2184-95.

Western blot detection of photosynthetic proteins from diatoms

western blot detection of photosynthetic proteins in diatoms

RbcL - T. punctigera, PetC - T. guillardii, PsbA - C. wailesii, PsbD - T. guillardii,

1 µg of total protein from various diatom cell pellets extracted with LiDS protein extraction buffer PEB (AgriSera) were separated for 40 min on 4-20% gradient Invitrogen NuPAGE gels and blotted 1h to PVDF. Blots were blocked with ECL Advance blocking agent for 1h at room temperature (RT) with agitation. Blot was incubated in the primary antibody at a dilution of 1: 10 000 to 1:25 000 (depending upon the antibody) for 1h at RT with agitation. The antibody solution was decanted and the blot was rinsed briefly twice, then washed once for 15 min and 3 times for 5 min in TBS-T at RT with agitation. Blot was incubated in secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated, from Agrisera, AS09 602 ) diluted to 1:25 000 in blocking buffer for 1h at RT with agitation. The blot was washed as above and developed for 5 min with ECL Advance according to the manufacturers instructions. Exposure time was 30-60 seconds with a BioRad VersaDoc CCD Imager.

High dilutions of primary and secondary antibodies are used to extend the pseudo-linear range of signal:target, by limiting steric interference which can lower signal:target ratio under excess antibody levels.

Protein Extraction – Diatoms

Although we’ve started incorporating an automatic, mechanical “bead-beater” in the lab for our samples (so fast! so consistent! so expensive!), every once in a while we find it necessary to go back to our tried and true “soni-thaw” method (sonication tip submerged into flash frozen samples until they reach the consistency of a green slushie). In both instances we always do a test run with a new species, where cells are exposed to increasing levels of disruption (ex: 1x-8x 30 second runs on the bead-beater or 1x-4x soni-thaw rounds). The samples are assayed for total protein quantity and then verified by Western Blotting for degradation. It sounds like a long process, but with practice you can get the ideal disruption protocol - that maximizes protein release while minimizing degradation - in a day and a half.

When growing pure cultures, we often just pellet the material by centrifugation, sometimes with the addition of the flocking agent Pluronic Acid F-68. When using mixed media samples (where ideal centrifugation values differ between cell types) or environmental samples (where a centrifuge is often a missing luxury) we resort to vacuum filtration of liquid onto binder-free (BF) filters. The binding agents used in the glass filters tend to gum up mechanical disruption methods, so it is vital to order the correct, binder-free filter for downstream protein extraction.