1 The Barshop Institute for Longevity and Aging Studies, University of Texas Health Science Center at San Antonio (UTHSCSA), San Antonio, USA
We have previously described use of a Balch homogenizer for the rapid, cost-effective and freeze-thaw independent homogenization of Caenorhabditis elegans (Bhaskaran, S., et al., 2011). This tool operates through shear forces generated by placing a ball bearing of known diameter within a confined chamber and then using two disposable syringes to manually advance worm samples past the micron sized gap that is formed between the chamber wall and the bearing. Using this tool we showed that worms of any developmental stage could be equally well homogenized after selecting an appropriately sized bearing to precisely control the wall-to-bearing clearance. Here we describe the use of the Balch Homogenizer for isolation of nuclei from C. elegans.
Since the primary goal of this study was to identify conditions that permitted rapid, freeze-thaw independent isolation of structurally-sound nuclei from any stage of worm development, our choice of buffers as well as centrifuge spin speeds to pellet nuclei were the same as those described in the embryonic and oocyte nuclei purification protocol of Mains and McGhee (1999). In their method, several approaches to disrupt worm samples were described, including use of a vintage motorized Stansted Cell Disruptor, a French Press, sonication and a mortar & pestle. Each method had its associated limitations, ranging from cost to sample heating. We established the following protocol for nuclei extraction which is rapid, does not require prior sample freezing, and can be applied equally to all stages of nematode development. Samples of nuclei collected using this protocol have been utilized in a number of downstream applications, including preparation of nuclear extracts for use in gel shift assays.
– 2x Nuclear Preparation Buffer (2xNPB): 20 mM HEPES (pH 7.6), 20 mM KCl, 3 mM MgCl2, 2 mM EGTA, 0.5 M sucrose, 1 M dithiothreitol, 1 mM PMSF, 20 μM E-64, (pre-chill to 4°C)
– Nuclear Extraction Buffer (NEB): 20 mM HEPES (pH7.6), 350 mM NaCl, 2 mM EDTA, 25% glycerol, 0.5 mM dithiothreitol, 0.5 mM PMSF, 10 μM E-64, (pre-chill to 4°C)
– Phosphate Buffered Saline (PBS), pH 7.4 (keep at room temperature)
– Balch Homogenizer (Isobiotec, Heidelberg, Germany), including 12 & 18 μm ball bearings. Refer to (Bhaskaran, S., et al., 2011) for full specification details of the homogenizer. (Pre-chill to 4°C)
– Bench top centrifuges
– SD1084 [gaIs148 [Pges-1::FLAG::PAB-1 + sur-5::GFP] (Pauli, F., et al., 2006)
For the following protocol we utilized a mixed-stage population of SD1084 worms containing a sur-5::GFP reporter gene. Use of this strain allowed us to monitor the degree of disruption of the nuclear membrane at each step of the protocol, as SUR-5::GFP is soluble. We have previously published ball bearing clearances required to either fracture or homogenize synchronized populations of worms at any stage of larval development [refer to Table 1 in (Bhaskaran, S., et al., 2011)]. The following protocol can therefore be easily modified to accommodate worms of any size. Nuclei in C. elegans range from ~1.8 – 7μm (Chen, L. et al., 2013).
- Wash mixed-stage, SD1084 worms off 5 x 10cm NGM plates using 5 ml PBS/plate. Pool, then pellet worms (800 x g, 3 minutes, RT), wash and re-pellet thrice more (3 x 15 mls PBS, RT). Transfer worms to ice (4°C). (Note: At this point we have also snap frozen worms in liquid nitrogen for later nuclei extraction. Freezing risks fracturing the nuclear membrane.)
- Add an equal volume of ice-cold 2xNPB to the worm pellet.
- Homogenize worms using a pre-chilled Balch Homogenizer (30 strokes /18 μm ball bearing, followed by 25 strokes / 12 μm ball bearing.)
- Transfer homogenate into a 15 ml falcon tube. Rinse the Balch Homogenizer with 2 ml of 1xNPB and combine with the first homogenate. Pellet nuclei (4000 x g, 5 minutes, 4°C). (Note: The supernatant corresponds to a crude cytoplasmic extract. To clarify, spin for 30 minutes at 35,000 x g at 4° Take the resulting supernatant, avoid the white flocculant, add glycerol to 25%, then snap freeze.)
- Resuspend nuclei in 5 ml 1xNPB containing 0.25% NP-40 + 0.1% Triton-X-100. To remove any large debris, centrifuge sample at 100 x g, 5 minutes, 4° Transfer supernatant containing nuclei to a fresh tube. (Note: NP-40 and Triton-X-100 are harsh detergents that will ultimately dissolve the nuclear membrane. If used at higher concentrations, or for longer periods, they will also disrupt protein structure. Use Tween-20, Saponin or Digitonin (0.2% – 0.5%, up to 30 min.) for milder membrane disruption.)
- To recover nuclei trapped within large debris, wash the low speed pellet twice with 5 ml 1xNPB containing 0.25% NP-40 + 0.1% Triton-X-100.
- Combine all supernatants and centrifuge at 4000 x g, 5 minutes, 4° Resuspend the pellet in 5 ml 1xNPB sans detergents. (Note: This sample represents the nuclei-enriched fraction.)
- To prepare a nuclear extract, re-pellet the nuclei (4000 x g, 5 minutes, 4°C).
- Resuspend the pellet in 2-4 volumes of NEB. Transfer to a microfuge tube, extract by gentle rotation 45 minutes at 4°
- Centrifuge at 10,000 x g for 14 minutes at 4°C to pellet debris.
- Carefully remove the supernatant and store in aliquots at -80°C (Note: This sample represents a crude nuclear extract positive for GFP (Fig. 1A). Surprisingly it also contains buoyant structures that stain positive with propidium iodide (Fig. 1B). These structures are chromatin skeletons (Fig. 1C) and they presumably avoid precipitation, in part because of the high glycerol content of the extraction buffer.)
Bhaskaran S, Butler JA, Becerra S, Fassio V, Girotti M, Rea SL (2011). Breaking Caenorhabditis elegans the easy way using the Balch homogenizer: an old tool for a new application. Analytical Biochemistry. 413, 123-132