An optimized protocol for isolation of soluble proteins from microalgae for two-dimensional gel electrophoresis analysis

Two-dimensional gel electrophoresis (2-DE)is a core proteomic technique to studyprotein expression and function in livingorganisms. Although it has been extensivelyused for investigation of bacterial, yeast,animal and plant tissue cells, there islimited information about the use of 2-DEin microalgal research. In this study, anumber of key chemical reagents, includingacetone, trichloroacetic acid, urea,thiourea, dithiothreitol, and tributylphosphine, were quantitatively evaluatedfor 2-DE of green microalgae, using Haematococcus pluvialis as a model system.The goal was to maximize the number andstaining intensity of protein spots whileminimizing streaking and smearing on thesecond dimensional SDS gel. Compared tonon-frozen immobilized pH gradients (IPG)strips, freezing of the IPG strips at –20 ^°C after isoelectric focusing (IEF)enhanced protein resolubilization andtransfer into the SDS gel, and thusimproved resolution while eliminatingvertical point streaking on the SDS gel. Itwas also confirmed that manipulation ofsample loading capacity is a simple,effective purification strategy forselective investigation of the proteins ofinterest and of varying abundances. Theprotocol was also successfully applied toprofiling protein expression in H.pluvialis under external stressconditions, indicating its potentialusefulness in further proteomics studies ofthis organism and related species.