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Fig. 3 | Parasites & Vectors

Fig. 3

From: Role of inhibitors of serine peptidases in protecting Leishmania donovani against the hydrolytic peptidases of sand fly midgut

Fig. 3

In vitro interactions of purified rLdISPs with trypsin and chymotrypsin. E. coli (BL-21) expressing the rLdISP1 and rLdISP2 proteins were purified through Ni2+/NTA column and confirmed by Western blot using anti-His primary antibody. a, b Western blot analysis of the expressed rLdISP1 and rLdISP2 proteins in BL-21 cells, respectively: Lane 1: whole cell lysate of cells (no insert); Lane 2: whole cell lysate of the uninduced cells; Lane 3: whole cell lysate of cells induced at 37 °C (0.75 mM IPTG); Lane 4: sup fraction of the induced cells; Lane 5: purified protein of rLdISP1 or rLdISP2. Interaction of rLdISP1 and rLdISP2 with trypsin (c, e) and chymotrypsin (d, f) were performed by co-immunoprecipitation. Trypsin or chymotrypsin antibody was bound to the activated resin and incubated with trypsin or chymotrypsin respectively. The immobilized antibody was further incubated individually with rLdISP1 or rLdISP2. Co-immunoprecipitate was collected and analysed by immunoblot, developed separately with anti-trypsin, anti-chymotrypsin, anti-ISP1 and anti-ISP2 antibodies. c-f Lane 1: negative control; Lane 2: flow through; Lane 3: wash fraction; Lanes 4–6: elution 1, elution 2, elution 3, respectively; Lane 7: trypsin or chymotrypsin as a positive control; Lane 8: rLdISP1 or rLdISP2 as a positive control

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