Development of an in-house protocol for the OFFGEL fractionation of plant proteins
Protein sample prefractionation using off-gel technology coupled to mass spectrometry-based proteomics has proven very efficient in identifying novel proteins of low abundance in various biological systems with the exception of plant tissues. In this study, we have prefractionated protein samples from wheat leaves and apoplastic fluid using the Agilent 3100 OFFGEL fractionator along 3-10 pH range into 24 fractions. Two methods were compared: 1/ the Agilent method based on the use of a proprietary starter kit which comprises all the necessary consumables, and 2/ an in-house method in which focusing solutions were made using electrophoresis grade chemicals, and immobilised pH gradient (IPG) strips and electrode pads were acquired elsewhere. Method comparisons and quality of focusing were assessed using pH distributions, one- and two-dimensional patterns. Both methods produced excellent electrophoretic patterns, with generally more better-resolved bands/spots observed with the in-house method. This study will allow other laboratories using OFFGEL device to consider in-house solutions.