Plant plasma membrane protein extraction and solubilization for proteomic analysis.
The plasma membrane (PM) exists as the interface between the cytosol and the environment in all living cells and is one of the most complex and differentiated membrane. The identification and characterization of membrane proteins (either extrinsic or intrinsic) is a crucial challenge since many of these proteins are involved in essential cellular functions such as cell signaling, osmoregulation, nutrition, and metabolism. Methods to isolate PM fractions vary according to organisms, tissues, and cell type. This chapter emphasizes isolation, from the model plant Arabidopsis thaliana, of PM fractions from a microsomal membrane fraction by two-phase partitioning, a methodology that utilizes the different surface properties of membranes. PM proteins that do not span the lipid bilayer are generally well recovered after 2D gel electrophoresis. By contrast, the recovery of transmembrane proteins requires first the depletion of the PM fraction from soluble proteins, being either cytosolic contaminants or functionally associated proteins, and second, to the use of specific solubilization procedures. This chapter presents protocols to strip PM based on alkaline treatment of membranes and to solubilize hydrophobic proteins to increase their recovery on 2D gels. Aquaporins that are highly hydrophobic proteins are used to probe the relevance of the procedures.
Santoni
Véronique
V
Biochimie et Physiologie Moléculaire des Plantes, INRA, Montpellier, France.
D020346:Aquaporins / Q000032:analysis; D017360:Arabidopsis / Q000737:chemistry*; D002021:Buffers; D002458:Cell Fractionation / Q000295:instrumentation / Q000379:methods; D002462:Cell Membrane / Q000737:chemistry*; D002478:Cells, Cultured; D015180:Electrophoresis, Gel, Two-Dimensional; D057927:Hydrophobic and Hydrophilic Interactions; D007202:Indicators and Reagents; D010940:Plant Proteins / Q000737:chemistry / Q000302:isolation & purification*; D040901:Proteomics / Q000295:instrumentation / Q000379:methods*; D012995:Solubility
D020346:Aquaporins; D002021:Buffers; D007202:Indicators and Reagents; D010940:Plant Proteins