Combined cup loading, bis(2-hydroxyethyl)disulfide, and protein precipitation protocols to improve the alkaline proteome of Lactobacillus hilgardii

Despite the large number of papers dealing with bacterial proteomes, very few include information about proteins with alkaline isoelectric points, because of the limits inherent in 2DE technology. Nonetheless, analyses of in silico proteomes of many prokaryotes show a bimodal distribution of their proteins based on their pIs, the most crowded areas lying between pI 4-7 and pI 9-11. The aim of the present research was to set up a general, simple and standardizable 2DE protocol suitable for studying alkaline proteome of L. hilgardii, a Gram-positive bacillus isolated from wine. The method has been also tested on a Gram-negative bacterium able to degrade aromatic pollutants, A. radioresistens S13. Optimization of the method was mainly focused on improving protein extraction and IEF (pI 6-11) separation protocols. Concerning IEF, different methods for sample loading (in-gel rehydration and cup-loading), and different reducing agents (DTT and HED) were tested and compared. The proposed protocol was found to resolve alkaline proteins from both our Lactobacillus and Acinetobacter strains efficiently, in spite of their different external layers, thus enabling more .......................