Comparison of different methods for the isolation of Arabidopsis thaliana nuclear membranes

Abstract

In animal and yeast system, subcellular fractionation has been widely used in studies of protein localization and organelle proteomics. Alternatively, it has not been an effective way of study in plant system because of some experimental limitations. The main aim of this study is to optimize subcellular and subnuclear fractionation of wild-type of Arabidopsis thaliana ecotype Colombia (Col-0) leaves by comparing three different methods to isolate nuclear membrane. In the study, we at first optimized nuclear washing steps to remove chloroplast contents from nuclear fractions. By optimizing speed of centrifugation and chemical component of the nuclear washing buffer, purified nuclear fractions was obtained. After measurement of protein amount for each fraction, purity of the fractions was analyzed by western blot assay with some specific cell compartment markers such as anti-Histone3 for nuclear fraction and anti-Rubisco for cytoplasmic fraction. Also, lactate dehydrogenase enzyme assay was used to confirm purity of the fractions. Then, subnuclear fractionation was done to isolate purified nuclear membrane. Three different methods were used to separate the nuclear membrane from whole purified nucleus. Nuclear compartment markers such as anti-Histone3 and anti-Fibrillarin was used in this step. In the study, Inner Nuclear Membrane protein (AtSUN2) was used as a nuclear membrane marker. All things considered, we conclude that the method with DNase digestion and high centrifugation speed (first method ) is a more effective way in separation of nuclear membrane fractions because the low centrifugal speed (second method) does not appear to be sufficient for separating the nuclear membrane, and the third method does not seem to be a very effective way as it requires a high centrifugal speed.