Extraction of plant proteins using typical extraction buffers leaves insoluble debris that cannot be investigated by conventional 2-DE technologies. In this paper, we present a scalable, off-line procedure for extraction of Arabidopsis thaliana homogenates that can be used in combination with both in-gel digestion and mass spectrometry. Based on sequential NaCl gradients and strong detergent fractionation, this new strategy allowed detection of 11 novel proteins from Arabidopsis thaliana that were altered in response to chilling stress.