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Establishing a liquid‐phase IEF in combination with 2‐DE for the analysis of Leishmania proteins
Author(s) -
Brobey Reynolds K. B.,
Soong Lynn
Publication year - 2007
Publication title -
proteomics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.26
H-Index - 167
eISSN - 1615-9861
pISSN - 1615-9853
DOI - 10.1002/pmic.200600587
Subject(s) - proteome , leishmania , computational biology , biology , proteomics , leishmania infantum , biochemistry , leishmaniasis , genetics , visceral leishmaniasis , parasite hosting , computer science , gene , world wide web
Abstract The recent completion of genome sequencing projects for Leishmania major and near completion for two other species, L. infantum and L. braziliensis , has provided the needed genomic information for investigating the proteomes of Leishmania parasites. However, the design of effective 2‐DE‐based proteome mapping for complex protozoan parasites like Leishmania has proven to be severely compromised due to extensive overcrowding of spots especially in the acidic regions, coupled to a relatively low representation of basic proteins. In the present study, we optimized a liquid‐phase IEF in combination with 2‐DE for L. amazonensis promastigote as a way of reducing protein complexity and enhancing representation for low‐abundance proteins on gels. Of 20 pH‐based fractions eluted from Rotofor™ cells, 5 representative fractions selected from acidic, basic or neutral regions of the proteome and with adequate protein concentration were further analyzed by 2‐DE using medium‐range IPG strips. On this basis, we were able to generate high‐resolution 2‐DE maps encompassing both the acidic and basic ends of the proteome with enhanced spot representation.