Conventional Gradient Centrifugation Research Articles

Rapid Affinity Purification of Tagged Plant Mitochondria (Mito-AP) for Metabolome and Proteome Analyses.

The isolation of organelles facilitates the focused analysis of subcellular protein and metabolite pools. Here we present a technique for the affinity purification of plant mitochondria (Mito-AP). The stable ectopic expression of a mitochondrial outer membrane protein fused to a GFP:Strep tag in Arabidopsis (Arabidopsis thaliana) exclusively decorates mitochondria, enabling their selective affinity purification using magnetic beads coated with Strep-Tactin. With Mito-AP, intact mitochondria from 0.5 g plant material were highly enriched in 30-60 min, considerably faster than with conventional gradient centrifugation. Combining gradient centrifugation and Mito-AP techniques resulted in high purity of >90% mitochondrial proteins in the lysate. Mito-AP supports mitochondrial proteome analysis by shotgun proteomics. The relative abundances of proteins from distinct mitochondrial isolation methods were correlated. A cluster of 619 proteins was consistently enriched by all methods. Among these were several proteins that lack subcellular localization data or that are currently assigned to other compartments. Mito-AP is also compatible with mitochondrial metabolome analysis by triple-quadrupole and orbitrap mass spectrometry. Mito-AP preparations showed a strong enrichment with typical mitochondrial lipids like cardiolipins and demonstrated the presence of several ubiquinones in Arabidopsis mitochondria. Affinity purification of organelles is a powerful tool for reaching higher spatial and temporal resolution for the analysis of metabolomic and proteomic dynamics within subcellular compartments. Mito-AP is small scale, rapid, economic, and potentially applicable to any organelle or to organelle subpopulations.

Mito-magneto: a tool for nanoparticle mediated mitochondria isolation.

The field of intracellular organelle targeting using nanoparticle (NP) is mushrooming rapidly. Thus, the area of nanotechnology-enabled targeting of mitochondrion, the cellular powerhouse, for diseases characterized by mitochondrial dysfunctions such as cancer, diseases of the central nervous system, and cardiovascular diseases is also growing at a rapid pace. Optimization of a NP's ability to target the mitochondria requires quantification of the particles in this subcellular organelle and isolation of mitochondria from the cells. Conventional gradient centrifugation used in currently available methods may not be appropriate for NP containing mitochondria isolation as these particles undergo Brownian motion under centrifugal forces yielding irreproducible results. There is only one method for centrifugation-free mitochondria isolation; however, this method requires immunoprecipitation. Thus, a reliable centrifugation and immunoprecipitation free method is urgently needed to support this growing field of nanotechnology-based mitochondria targeting. Here, we report a mitochondria-targeted magnetic NP, Mito-magneto, to avoid centrifugation and immunoprecipitation methods for isolation of functional, respiration active pure mitochondria, which can be used to analyze and quantify mitochondria targeting properties of various NPs as an important tool for the growing field of "mitochondrial nanomedicine".

Mesenchymal Stem Cell Lines Isolated by Different Isolation Methods Show Variations in the Regulation of Graft-versus-host Disease

Since the discovery of the immunomodulation property of mesenchymal stem cells (MSCs) about a decade ago, it has been extensively investigated whether MSCs can be used for the treatment of immune-related diseases, such as graft-versus-host disease (GvHD). However, how to evaluate the efficacy of human MSCs for the clinical trial is still unclear. We used an MHC-mismatched model of GvHD (B6 into BALB/c). Surprisingly, the administration of the human MSCs (hMSCs) could reduce the GvHD-related mortality of the mouse recipients and xenogeneically inhibit mouse T-cell proliferation and IFN-γ production in vitro. We recently established a new protocol for the isolation of a homogeneous population of MSCs called subfractionation culturing methods (SCM), and established a library of clonal MSC lines. Therefore, we also investigated whether MSCs isolated by the conventional gradient centrifugation method (GCM) and SCM show different efficacy in vivo. Intriguingly, clonal hMSCs (hcMSCs) isolated by SCM showed better efficacy than hMSCs isolated by GCM. Based on these results, the MHC-mismatched model of GvHD may be useful for evaluating the efficacy of human MSCs before the clinical trial. The results of this study suggest that different MSC lines may show different efficacy in vivo and in vitro.

Characterization of mouse clonal mesenchymal stem cell lines established by subfractionation culturing method

To characterize single-cell-derived mouse clonal mesenchymal stem cells (mcMSCs) established with bone marrow samples from three different mouse strains. We established mcMSC lines using subfractionation culturing method from bone marrow samples obtained from long bones. These lines were characterized by measuring cell growth, cell surface epitopes, differentiation potential, lineage-specific gene expression and T-cell suppression capability. Nonclonal MSCs isolated by the conventional gradient centrifugation method were used as controls. All mcMSC lines showed typical nonclonal MSC-like spindle shape morphology. Lines differed in optimal growth density requirement. Cell surface epitope profiles of these mcMSC lines were similar to those of nonclonal MSCs. However, some lines exhibited different expression levels in a few epitopes, such as CD44 and CD105. Differentiation assays showed that 90% of the mcMSC lines were capable of differentiating into adipogenic and/or chondrogenic lineages, but only 20% showed osteogenic lineage differentiation. T-cell suppression analysis showed that 75% of the lines exhibited T-cell suppression capability. mcMSC lines have similar cell morphology and cell growth rate but exhibit variations in their cell surface epitopes, differentiation potential, lineage-specific gene expression and T-cell suppression capability.

The quaternary structures of SV40 large T antigen and tumor suppressor p53: analysis by gel electrophoresis.

Protein-protein interactions have become a major theme in molecular biology. In many cases, the homo-oligomeric structure of a protein is essential for the activity of that protein. For example, both SV40 large T antigen and the cellular tumor suppressor p53 have distinctive quaternary structures that determine their intrinsic functions. In other cases, hetero-oligomeric structures lead to the gain or loss of function of the interacting proteins. Again, T antigen and p53 are excellent examples of this kind of interaction. T antigen-binding blocks the function of p53 in the regulation of cellular proliferation (1–3), and p53 in turn may modulate T antigen’s replication functions (4–6). In view of the obvious importance of protein-protein interactions, simple techniques for the analysis of protein oligomers will be crucial to an understanding of their functions. Here, we review the use of gradient gel electrophoresis and protein crosslinking to analyze the quaternary structures of SV40 large T antigen, murine p53, and their complexes. This technique offers several advantages over conventional gradient centrifugation for analyzing quaternary structure. Multiple samples can be examined in parallel with resolution superior to that of gradient centrifugation. We have used purified proteins produced by a baculovirus expression system. The methods for these studies have been described previously (7,8).

Application of a microseparation technique allowing for extensive marker studies on small bone marrow specimens

The characterization of surface and intracellular markers of hematopoietic cells by immunocytochemical methods is widely used. While examination of cells of the peripheral blood can easily be performed, investigations of bone marrow cells can be hampered by technical difficulties in labeling routine marrow smears or by limited size of specimens required for cytocentrifugation. A simple and highly efficient method for isolation of bone marrow cells by a microseparation technique is described. Only two to three drops of a routine bone marrow aspirate are necessary for preparation of up to 100 cytocentrifuge slides thereby exceeding separation efficiency of conventional gradient centrifugation by three- to more than four-fold. The application of the method for marker studies on normal marrow and marrow from patients with hematologic malignancies is exemplified.

The use of conventional and zonal centrifugation to study the life cycle of mammalian cells. Phospholipid and macromolecular synthesis in neoplastic mast cells.

1. Conventional gradient centrifugation has been used to separate cells according to their position in the cell cycle, and to obtain synchronously growing cells. Analysis of prelabelled cells by gradient centrifugation confirms that phospholipid, protein and RNA synthesis is continuous throughout the cell cycle and shows that the rate of synthesis begins to increase already during the G(1) phase. The pattern of phospholipid degradation follows that of synthesis. 2. The limitations of conventional gradient centrifugation have been overcome by use of a zonal rotor. Analysis of prelabelled cells confirms the results obtained by conventional centrifugation and in addition shows that the rates of phospholipid, protein and RNA synthesis decrease during the G(2) phase. The mean cell volume and the net amount of phospholipid, protein and RNA, unlike that of DNA, are found to increase continuously throughout the intermitotic period. 3. These results show that the synthesis of macromolecules, and probably that of membranes also, is controlled by a mechanism other than that of gene dosage.