An improved silver staining procedure for schizodeme analysis in polyacrylamide gradient gels

Gonçalves, A. Pedro; Nehme, N. S.; Morel, Carlos Médicis

doi:10.1590/s0074-02761990000100017

Cited by 18 publications

(7 citation statements)

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“…The samples were denatured prior to analysis by adding one μL of 0.5 M NaOH/10 mMEDTA and then electrophoresed for 3 h at 10 V/cm using 0.5 × TBE buffer (pH8.4). SSCP patterns were visualized by silver staining as described elsewhere [ 30 ].…”

Section: Methodsmentioning

confidence: 99%

Evaluation of the genetic polymorphism of Plasmodium falciparum P126 protein (SERA or SERP) and its influence on naturally acquired specific antibody responses in malaria-infected individuals living in the Brazilian Amazon

Pratt-Riccio

Sallenave-Sales

Oliveira-Ferreira

et al. 2008

Malar J

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Background: The Plasmodium falciparum P126 protein is an asexual blood-stage malaria vaccine candidate antigen. Antibodies against P126 are able to inhibit parasite growth in vitro, and a major parasite-inhibitory epitope has been recently mapped to its 47 kDa N-terminal extremity (octamer repeat domain -OR domain). The OR domain basically consists of six octamer units, but variation in the sequence and number of repeat units may appear in different alleles. The aim of the present study was to investigate the polymorphism of P126 N-terminal region OR domain in P. falciparum isolates from two Brazilian malaria endemic areas and its impact on anti-OR naturally acquired antibodies.

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Section: Methodsmentioning

confidence: 99%

Evaluation of the genetic polymorphism of Plasmodium falciparum P126 protein (SERA or SERP) and its influence on naturally acquired specific antibody responses in malaria-infected individuals living in the Brazilian Amazon

Pratt-Riccio

Sallenave-Sales

Oliveira-Ferreira

et al. 2008

Malar J

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“…The digestion products were electrophoresed on a 4.5-10% polyacrylamide gradient gel and stained with silver nitrate. 11 The quantification of common bands was per- formed with the formula C/A ϩ B ϩ C, where C is the number of bands common to the two stocks being compared, and B and A correspond to bands present only on the first and second stocks, respectively.…”

Section: Trypanosoma Cruzi Stocksmentioning

confidence: 99%

Genetic structure and phylogenetic relationships of Colombian Trypanosoma cruzi populations as determined by schizodeme and isoenzyme markers.

Jaramillo

Moreno²,

Triana³

et al. 1999

The American Journal of Tropical Medicine and Hygiene

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Abstract. Twenty-four Trypanosoma cruzi stocks isolated from vectors and from human and Didelphis marsupialis hosts from highly separated sylvatic localities in Colombia were characterized by isoenzyme and schizodeme analyses. The stocks were collected primarily from sylvatic ecotopes representing areas of low, moderate, and high endemicity for Chagas' diseases in Colombia. Parasites were characterized mainly by schizodeme analysis with the restriction enzyme Eco RI and the isoenzyme analysis was performed at 10 genetic loci. These analyses demonstrated an agreement between the classifications based on the isoenzyme analysis and on the restriction fragment length polymorphism patterns obtained with the Colombian stocks. There is clear evidence of demic subdivision between the eastern (E) and western (W) stocks separated by the Andean Mountains and Magdalena River, which is likely due to the geographic isolation generated by these topographic features. Heterozygosity estimates indicate that the E group could be more ancient than the W group. As was postulated in a previous study, these results are also compatible with the existence of a clonal population structure in Colombian sylvatic T. cruzi. Evidence presented here failed to demonstrate a correlation between the degree of endemy and genetic clustering. Finally, schizodeme and isoenzymatic analyses comparing Colombian T. cruzi stocks with others from Chile confirm that Colombian isolates are genetically related to zymodeme 1 and distant from zymodeme 2.

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“…Soluble proteins (40 μg) were resolved using 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and silver staining, as previously described [29,30]. Electrophoresis was performed at 25°C in a Mini Protean II system (BioRad Laboratories, US).…”

Section: Methodsmentioning

confidence: 99%

Participation of heparin binding proteins from the surface of Leishmania (Viannia) braziliensis promastigotes in the adhesion of parasites to Lutzomyia longipalpis cells (Lulo) in vitro

et al. 2012

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BackgroundLeishmania (V.) braziliensis is a causative agent of cutaneous leishmaniasis in Brazil. During the parasite life cycle, the promastigotes adhere to the gut of sandflies, to avoid being eliminated with the dejection. The Lulo cell line, derived from Lutzomyia longipalpis (Diptera: Psychodidae), is a suitable in vitro study model to understand the features of parasite adhesion. Here, we analyze the role of glycosaminoglycans (GAGs) from Lulo cells and proteins from the parasites in this event.MethodsFlagellar (Ff) and membrane (Mf) fractions from promastigotes were obtained by differential centrifugation and the purity of fractions confirmed by western blot assays, using specific antibodies for cellular compartments. Heparin-binding proteins (HBP) were isolated from both fractions using a HiTrap-Heparin column. In addition, binding of promastigotes to Lulo cells or to a heparin-coated surface was assessed by inhibition assays or surface plasmon resonance (SPR) analysis.ResultsThe success of promastigotes subcellular fractionation led to the obtainment of Ff and Mf proteins, both of which presented two main protein bands (65.0 and 55.0kDa) with affinity to heparin. The contribution of HBPs in the adherence of promastigotes to Lulo cells was assessed through competition assays, using HS or the purified HBPs fractions. All tested samples presented a measurable inhibition rate when compared to control adhesion rate (17 ± 2.0% of culture cells with adhered parasites): 30% (for HS 20μg/ml) and 16% (for HS 10μg/ml); HBP Mf (35.2% for 10μg/ml and 25.4% for 20μg/ml) and HBP Ff (10.0% for 10μg/ml and 31.4% for 20μg/ml). Additionally, to verify the presence of sulfated GAGs in Lulo cells surface and intracellular compartment, metabolic labeling with radioactive sulfate was performed, indicating the presence of an HS and chondroitin sulfate in both cell sections. The SPR analysis performed further confirmed the presence of GAGs ligands on L. (V.) braziliensis promastigote surfaces.ConclusionsThe data presented here point to evidences that HBPs present on the surface of L. (V.) braziliensis promastigotes participate in adhesion of these parasites to Lulo cells through HS participation.

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An improved silver staining procedure for schizodeme analysis in polyacrylamide gradient gels

Cited by 18 publications

References 0 publications

Evaluation of the genetic polymorphism of Plasmodium falciparum P126 protein (SERA or SERP) and its influence on naturally acquired specific antibody responses in malaria-infected individuals living in the Brazilian Amazon

Evaluation of the genetic polymorphism of Plasmodium falciparum P126 protein (SERA or SERP) and its influence on naturally acquired specific antibody responses in malaria-infected individuals living in the Brazilian Amazon

Genetic structure and phylogenetic relationships of Colombian Trypanosoma cruzi populations as determined by schizodeme and isoenzyme markers.

Participation of heparin binding proteins from the surface of Leishmania (Viannia) braziliensis promastigotes in the adhesion of parasites to Lutzomyia longipalpis cells (Lulo) in vitro

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