Evaluation of chloroform/methanol extraction to facilitate the study of membrane proteins of non-model plants.
Membrane proteins are of great interest to plant physiologists because of their important function in many physiological processes. However, their study is hampered by their low abundance and poor solubility in aqueous buffers. Proteomics studies of non-model plants are generally restricted to gel-based methods. Unfortunately, all gel-based techniques for membrane proteomics lack resolving power. Therefore, a very stringent enrichment method is needed before protein separation. In this study, protein extraction in a mixture of chloroform and methanol in combination with gel electrophoresis is evaluated as a method to study membrane proteins in non-model plants. Benefits as well as disadvantages of the method are discussed. To demonstrate the pitfalls of working with non-model plants and to give a proof of principle, the method was first applied to whole leaves of the model plant Arabidopsis. Subsequently, a comparison with proteins extracted from leaves of the non-model plant, banana, was made. To estimate the tissue and organelle specificity of the method, it was also applied on banana meristems. Abundant membrane or lipid-associated proteins could be identified in both tissues, with the leaf extract yielding a higher number of membrane proteins.
Vertommen
Annelies
A
Division of Crop Biotechnics, Department of Biosystems, K U Leuven, Leuven, Belgium. Annelies.vertommen@biw.kuleuven.be
Panis
Bart
B
Swennen
Rony
R
Carpentier
Sebastien Christian
SC
D002457:Cell Extracts; D005591:Chemical Fractionation / Q000379:methods*; D002725:Chloroform / Q000737:chemistry*; D015180:Electrophoresis, Gel, Two-Dimensional; D008565:Membrane Proteins / Q000302:isolation & purification* / Q000378:metabolism; D018519:Meristem / Q000378:metabolism; D000432:Methanol / Q000737:chemistry*; D008954:Models, Biological; D028521:Musa / Q000378:metabolism*; D018515:Plant Leaves / Q000378:metabolism; D010940:Plant Proteins / Q000302:isolation & purification* / Q000378:metabolism; D015203:Reproducibility of Results; D012995:Solubility
D002457:Cell Extracts; D008565:Membrane Proteins; D010940:Plant Proteins; D002725:Chloroform; D000432:Methanol