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32 Lahiri and Bhattacharyasurfaces such as those of cells. The aliquots of the Ultrastructural study of the flagellum bytreated cells were fixed with 2.5% glutaraldehyde transmission electron microscopy: Cells were fixed(Sigma) in 0.1M phosphate buffer (pH 7.2-7.4; Sigma) in a suitably buffered aldehyde fixative (2.5%for 24 h and washed in the same buffer for 15 min x 4. glutaraldehyde grade I; Sigma) in 0.1 M sodiumThe samples were rinsed thrice with double distilled cacodylate buffer (pH 7.4; Sigma) at 4° C for 1-4 h.water, each time for 5 min. Then they were dehydrated Then the cells were washed for 2 h or overnight at 4° Cin different grades of alcohol: 50, 70, 80, 90, 95 and in three changes of 0.1M sodium cacodylate buffer100% ethanol (20 min each). Gold coating of 200Å (pH 7.4). Cells were post-fixed in 1% OsO 4 (Sigma) inwas done at 5 mA using Giko Engineering-IB2 ion 0.8% potassium ferricyanide (Sigma) for 1-2 h at roomcoater. The cells were dried using vacuum pump and temperature and protected from light (Nakano et al.,observed under a SEM (model HITACHI S-530), and 2001). The above cells were washed for 5 min x 2 withthe photographs were taken by MAMIYA 6 x 7 camera distilled water. Dehydration was done using theusing NOVA 120 ASA films. following grades of alcohol: 50, 70, 90 and 95%PFR protein purification: To purify the proteinethanol, each for 15 min, and 100% ethanol for 15 mincomponent of PFR, it was necessary to remove thex 4. Finally, the treated cells were embedded in Eponflagellar membrane. The flagellar membranes werepolybed 820 epoxy resin. Ultrathin sections were cutremoved by non-ionic detergent treatment. Flagellarand stained with 5% aqueous uranyl acetate (Sigma)fractions of L. donovani promastigotes, obtained asand lead citrate (Sigma) and observed under adescribed above, were subjected to three rounds oftransmission electron microscope.treatment with 2% Nonidet P-40 (Sigma) in PBS(Sigma) at 0-4° C under constant shaking. Each 15 minRESULTSround of detergent treatment was followed by When observed under a SEM, it was revealed that thecentrifugation at 17,300 x g for 20 min at 4° C cells centrifuged at 2600 x g retained their flagella(Moreira-Leite et al., 1999). The pellet of the final (Fig. 1). The flagella were detached from the intact cellcentrifugation step was dissolved in PBS (Sigma) and bodies when centrifugation was carried out at 3200 x gsubjected to a brief treatment with 0.0015% trypsin (Fig. 2). However, the cells were ruptured when(type XIII, TPCK-treated, Sigma) for 90 s at 28° C. centrifuged at 3700 x g (Fig. 3).The trypsin treatment was stopped by adding 20-foldSDS-PAGE analysis showed two protein bands of molexcess soyabean trypsin inhibitor (Sigma). Thewt 76 kDa and 68 kDa that are presumed to be tworesultant protein fractions were subjected to SDSfractionsof the PFR proteins (Fig. 4).PAGE.SDS-PAGE: This method is based on the separation ofproteins according to mol wt and is particularly usefulfor monitoring protein purification. The sample to berun on SDS-PAGE was mixed with loading dye(protein:loading dye, 1:1) and boiled for 5 min in awater bath. The stock loading dye had the followingcomposition: double distilled water, 4.8 ml; TRIS (pH6.8), 1.2 ml; 10% SDS (Sigma), 2 ml; glycerol(Sigma), 1 ml and bromophenol blue (Sigma), 0.5 ml.Before use, 950 µl of stock solution was mixed with 50µl of β-mercaptoethanol. The sample was runsimultaneously with protein markers (Broad Range,Bangalore GENEI, Bangalore, India) in differentwells at a constant voltage of 100 V in 10% resolvinggel and 4% stacking gel. The gel was then fixed inmethanol and stained with Coomassie Brilliant BlueR-250 (Sigma) for a few h, and then washed indestaining solution until clear bands were visible(Laemmli, 1970).Transmission electron microscopy study revealed theultrastructural details of PFR. The longitudinalsection of a flagellum shows that the lattice-like PFRruns parallel to the axoneme even before theemergence of the flagellum from the flagellar pocket0002 15KV 5umFig. 1: SEM of a L. donovani cell centrifuged at 2600 x g.