title: A general method for the extraction of citrus leaf proteins and separation by 2D electrophoresis: a follow up.
abstract: With the aim of studying differentially expressed proteins as a function of abiotic and biotic stress in citrus plants, we optimized a protocol for the extraction of total leaf proteins and their 2-DE separation using commercially available immobilized pH gradient strips (IPGs) in the first dimension. Critical factors for good reproducibility of citrus leaf protein separation were identified: trichloroacetic acid (TCA)/acetone precipitation after extraction in lysis buffer, sample fractionation on narrow range overlapping IPGs and sample-cup loading at the anodic or cathodic end of the strip. The use of thiourea and a strong detergent (C7BzO) in the solubilization/rehydration buffer, coupled with the increase to 10% of SDS in the equilibration buffer before the second dimension seemed to affect positively the resolution of basic proteins. Using our protocol we resolved about 30 basic proteins on 6.3-8.3 pH range strips. Further, our protocol was successfully applied reproducibly on the analysis of control and salt exposed leaf samples of Citrus reshni Hort. Ex Tan.
authors.affiliation: National Research Council (CNR)-Institute of Biophysics (IBF), CNR Research Area, Via Moruzzi 1, 56124 Pisa, Italy. bianca.elena.maserti@pi.ibf.cnr.it
authors.forename: B E
authors.initials: BE
authors.lastname: Maserti
authors.forename: C M
authors.initials: CM
authors.lastname: Della Croce
authors.forename: F
authors.initials: F
authors.lastname: Luro
authors.forename: R
authors.initials: R
authors.lastname: Morillon
authors.forename: M
authors.initials: M
authors.lastname: Cini
authors.forename: L
authors.initials: L
authors.lastname: Caltavuturo
mesh_terms: D002957:Citrus / Q000378:metabolism*; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D006863:Hydrogen-Ion Concentration; D018515:Plant Leaves / Q000378:metabolism*; D010940:Plant Proteins / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification*; D015203:Reproducibility of Results
chemical_list: D010940:Plant Proteins