Cytoskeleton Fractionation:

Based on (Robinson et al., 1991:Methods Enzymol 196; pp. 285-99.)

1. Mid log phase trypanosomes are pellet at 1000 x g for 10 minutes and washed twice with PBS.

2. Cells are resuspended at 2.0 x 10⁸ cells / ml in PEME (100 mM PIPES, 2mM EGTA, 0.1 mM EDTA, 1 mM MgSO₄) supplemented with 1% Nonidet-P40 (NP-40) plus the protease inhibitors Aprotinin (5 mg/ml) and Leupeptin (5 mg/ml), and incubated at room temperature for 5 minutes.

3. A lysate sample (equivalent to 1/5 of the total volume) is removed, and the remaining suspension is spun at 1000 x g for 7 – 10 minutes.

4. The supernatant of the 1000 x g spin is removed (S1) and the pellet of the 1000 x g spin is washed twice in PEME, supplemented with 1% NP-40 plus protease inhibitors and resuspended in same volume as the S1 in PEME, 1% NP-40.

5. All fractions are either immediately frozen at –20 degrees or boiled for four minutes after adding 1/3 of the volume of each fraction of 4X Laemmli Sample Buffer [10% Glycerol, 4.5% SDS, 20% 4X upper tris-buffer (0.5 M tris, 0.4% SDS, pH 6.8), 0.03% bromophenol blue, 5% b-mercaptoethanol]. Extraction of the cytoskeleton can be visually monitored by light microscopy. For confocal images, a small aliquot a each fraction ( less than 4 ml ) is mixed 1:1 with VectishieldÔ mounting medium, and the cover slips are sealed with fingernail polish.

CaCl extraction of cytoskeletons to solubilize subpellicular microtubules.

Also based on (Robinson et al., 1991:Methods Enzymol 196; pp. 285-99.)

1. Cytoskeleton fractions (L, S1, P1) are prepared as described above, with the following modifications. The P1 is washed once in PEME, 1% NP-40 plus protease inhibitors, and once in PMN (10 mM NaPO₄ pH 7.4, 150 mM NaCl, 1 mM MgCl) supplemented with 1% NP-40 and protease inhibitors.

2. The P1 fraction is then resuspended in PMNCa (PMN, 3 mM CaCl₂, 1% NP-40, and protease inhibitors).

3. An aliquot P1 is taken and treated for SDS-PAGE or microscopy, and the remaining P1 is left on ice for 30 minutes to allow the sub-pellicular microtubules to depolymerize.

4. Following depolymerization, the sample is centrifuged at 16,000 x g for 10 minutes. The supernatant is removed (S2, subpellicular fraction), and the pellet is washed once in PMNCa, 1% NP-40 plus protease inhibitors, and this P2 (flagellar fraction) is resuspended to a volume equal to the S2 in PMNC 1% NP-40. All stages of fractionation are monitored visually by light microscopy prior to beginning the subsequent steps.