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. Author manuscript; available in PMC: 2011 Sep 1.
Published in final edited form as: Microbes Infect. 2010 May 11;12(10):716–726. doi: 10.1016/j.micinf.2010.04.014

Fig. 2. Verification of a trans-sialidase antibody and Interactions of TcPKAc with the members of trans-sialidase super family.

Fig. 2

A commercial anti- sialidases antibody (rabbit) was first tested and verified for its ability to recognize T. cruzi trans-sialidases recombinant proteins, including a purified active trans-sialidase and trans-sialidase fusion proteins in crude yeast lysates (primary antibody dilution 1:1000; secondary antibody dilution 1: 5000) (Fig.2A–B). Co-immunoprecitation was performed using Triton X-100 protein extracts from trypomastigotes and a trans-sialidase polyclonal antibody (anti-TCTS 6 11/2, mouse), a commercial sialidase antibody, a T. cruzi SA85-1.1polyclonal antibody (rabbit) and a TcPKAc mAb(antibody dilutions:1;100 for immunoprecipitation, 1:1000 for immunoblot)(Fig.2C–H).

A. The sialidase antibody binds to recombinant protein of TCTS 611/2.

Lane1, 100ug of BSA as negative control;

Lane 2, 100ng of recombinant protein of TCTS 611/2.

B. The sialidase antibody binds to fusion protein of trans-sialidase (Tc00.1047053509187.10) in yeast lysate.

Lane 1, 150 ug of wild type yeast lysate without transformation as negative control;

Lane 2, 150ug of yeast lysate with transformation of both bait and pAD- trans-sialidase (Tc00.104053509187.10) constructs. Antibody recognized the pAD- trans-sialidase protein.

C. Co-IP in Triton X −100 protein extracts from trypomastigotes with a TcPKAc mAb. Complex pulled down by a TcPKAc mAb contained proteins recognized by a commercial sialidase antibody on immunoblot.

Lane 1, Negative control using an unrelated mAb (anti-bag5);

Lane 2, Immunoprecipitation with TcPKAc mAb. There are four bands ranged from 85 kDa to 200 kDa, which reacted with this sialidase antibody.

D. Co-IP in Triton X −100 protein extracts from trypomastigotes with a commercial sialidase antibody. The sialidase antibody was able to pull down a protein, which reacted with a TcPKAc mAb on immunoblot.

Lane 1, negative control using pre-immune rabbit serum;

Lane 2, Immunoprecipitation with Trans-sialidase antibody. There is a 40-kDa TcPKAc protein band.

E. Co-IP in Triton X −100 protein extracts from trypomastigotes with a TcPKAc mAb.

Complex pulled down by a TcPKAc mAb contained proteins recognized by a SA85-1.1 antibody on immunoblot.

Lane 1, Negative control using an unrelated mAb (anti-bag5);

Lane 2, Immunoprecipitation with TcPKAc mAb. There is an 85 kDa SA85-1.1 protein band.

F. Co-IP in Triton X −100 protein extracts from trypomastigotes with a SA85-1.1 antibody.

Precipitated complex by a SA85-1.1 antibody contained TcPKAc.

Lane 1, Negative control using pre-immune rabbit serum;

Lane 2, Immunoprecipitation with SA85-1.1 antibody. There is a 40-kDa TcPKAc protein band.

G. Co-IP in Triton X −100 protein extracts from trypomastigotes with a TcPKAc mAb. Complex pulled down by a TcPKAc mAb contained proteins recognized by a trans-sialidase antibody (anti-TCTS 6 11/2) on immunoblot.

Lane 1, Negative control using an unrelated mAb (anti-bag5);

Lane 2, Immunoprecipitation with TcPKAc mAb. There are bands with molecular weights from 85–200 kDa.

H. Co-IP in Triton X −100 protein extracts from trypomastigotes with a TCTS 6 11/2 antibody. Precipitated complex by a TCTS 6 11/2 antibody contained TcPKAc.

Lane 1, Negative control using pre-immune mouse serum;

Lane 2, Immunoprecipitation with Anti-TCTS 6 11/2;