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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 2 ml bead tubes containing MPBio lysing matrix D (SKU 116913050) and 700 μl 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 0.55 M TRIS buffer, 0.3 M LDS, 4.3 M glycerol and 2 mM EDTA.

The FastPrep was set to run at 6.5 m/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 min at 10 000 x g 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.

Agrisera's primary antibody colleaction for diatoms.

 



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 1 h 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 1 h 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 manufacturer's 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 have started incorporating an automatic, mechanical "bead-beater" in the lab for our samples (so fast! so consistent!), 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.

Sonication tips
Sonicate the cell suspension just until the sample thaws with the Sonicator microtip, at 30% duty cycle, continuous pulse, or select a setting at which the intensity is such that you do not froth the sample uncontrollably or cause it to leave the tube.
The microtip must be kept below the surface of the buffer or severe frothing will occur.
Test different settings on your sonicator to generate intense but controllable energy.
Take care not to damage the tube by pressing the tip hard against the plastic. The sonicator is capable of drilling a hole in a plastic tube. Refreeze the sample while holding the tube vertically with forceps and lowering it into liquid N2. At least 15 seconds in liquid N2. is required to freeze thoroughly. Note that the freezing is part of the breakage.
Repeat the sonication.
A total of two sonications is sufficient for many cell types and plant tissues.

However, certain cells require an additional cycle or two. To check for complete lysis, centrifuge the sample at full speed (10000 x g) for one minute and examine the pellet.

If the majority of the pellet is still an intense green color, additional sonication is required. Other tips: We do not generally sonicate with less than 200 µl or more than 700 µl of buffer. A 1.5 ml conical Eppendorf tube concentrates the energy in a smaller space than a larger or broader tube. Samples become extremely brittle when frozen in liquid N2, To avoid losing fragments at the start of sonication, we suggest leaving the sample for 30 - 60 seconds at room temp before sonicating.  

Note: chosen extraction conditions depend upon type of sample. The aim is that extraction should be carried out promptly to lower risk of degradation and consistently, especially if samples are for quantitation (bead beater). Algal samples can be collected by filtration on a polycarbonate or centrifugation (speed and time has to be adjusted depending upon the species). Test and chose the most optimal method.

Agrisera's primary antibody collection for diatoms.