3xmyc egfp omp25 mito ip control mito construct addgene  (Addgene inc)

Journal: Nature protocols

Article Title: Rapid immunopurification of mitochondria for metabolite profiling and absolute quantification of matrix metabolites

doi: 10.1038/nprot.2017.104

Figure Lengend Snippet: Step numbers in the figure correspond to those in the PROCEDURE of the manuscript. Cells expressing appropriate amounts of the 3XMyc-EGFP-OMP25 gene (Control-MITO cells) or the 3XHA-EGFP-OMP25 gene (HA-MITO cells) are generated using retroviral transduction and fluorescence-activated cell sorting in steps 1–13. Control-MITO or HA-MITO cells are quickly harvested and homogenized, with the homogenate precleared to remove cells, nuclei, and other large debris, resulting in a suspension of mitochondria and other organelles (steps 14–23). After a short anti-HA immunopurification (IP) (3.5 min in length) to capture HA-tagged mitochondria, antibody-conjugated beads are quickly washed three times (steps 24–29). The majority of the isolated mitochondria are extracted for metabolites and the corresponding mole quantities determined by liquid chromatography and mass spectrometry (LC/MS) (steps 30–50). The remaining isolated mitochondria are then lysed for protein and whole-cell equivalents of mitochondria in each IP sample are determined by immunoblot analysis (steps 30–37, 51–60). Confocal microscopy and volumetric analysis of HA-MITO cells is used to quantify the total mitochondrial volume per cell, which is then adjusted using the percentage of mitochondrial volume occupied by the matrix (~63.16% of mitochondrial volume = matrix)42 (steps 61–77). The matrix concentration of a mitochondrial metabolite is derived from the combination of all of these measurements (step 78).

Article Snippet: Plasmids carrying the 3XMyc-EGFP-OMP25 gene (Control-MITO construct, Addgene plasmid #83355) 21 and 3XHA-EGFP-OMP25 gene (HA-MITO construct, Addgene plasmid #83356) 21 .

Techniques: Expressing, Generated, Transduction, Fluorescence, FACS, Immu-Puri, Isolation, Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Western Blot, Confocal Microscopy, Concentration Assay, Derivative Assay

Journal: Nature protocols

Article Title: Rapid immunopurification of mitochondria for metabolite profiling and absolute quantification of matrix metabolites

doi: 10.1038/nprot.2017.104

Figure Lengend Snippet: Step numbers in the figure correspond to those in the PROCEDURE of the manuscript. Cells expressing appropriate amounts of the 3XMyc-EGFP-OMP25 gene (Control-MITO cells) or the 3XHA-EGFP-OMP25 gene (HA-MITO cells) are generated using retroviral transduction and fluorescence-activated cell sorting in steps 1–13. Control-MITO or HA-MITO cells are quickly harvested and homogenized, with the homogenate precleared to remove cells, nuclei, and other large debris, resulting in a suspension of mitochondria and other organelles (steps 14–23). After a short anti-HA immunopurification (IP) (3.5 min in length) to capture HA-tagged mitochondria, antibody-conjugated beads are quickly washed three times (steps 24–29). The majority of the isolated mitochondria are extracted for metabolites and the corresponding mole quantities determined by liquid chromatography and mass spectrometry (LC/MS) (steps 30–50). The remaining isolated mitochondria are then lysed for protein and whole-cell equivalents of mitochondria in each IP sample are determined by immunoblot analysis (steps 30–37, 51–60). Confocal microscopy and volumetric analysis of HA-MITO cells is used to quantify the total mitochondrial volume per cell, which is then adjusted using the percentage of mitochondrial volume occupied by the matrix (~63.16% of mitochondrial volume = matrix)42 (steps 61–77). The matrix concentration of a mitochondrial metabolite is derived from the combination of all of these measurements (step 78).

Article Snippet: Plasmids carrying the 3XMyc-EGFP-OMP25 gene (Control-MITO construct, Addgene plasmid #83355) 21 and 3XHA-EGFP-OMP25 gene (HA-MITO construct, Addgene plasmid #83356) 21 .

Techniques: Expressing, Generated, Transduction, Fluorescence, FACS, Immu-Puri, Isolation, Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Western Blot, Confocal Microscopy, Concentration Assay, Derivative Assay