Mitochondria were isolated from rosette leaves of four week-old transgenic Arabidopsis plants expressing LEA38-GFP in mitochondria [25 (link)]. Leaves were grinded with a Waring blender in 10 mL isolation buffer (30 mM MOPS pH 7.8, 330 mM sorbitol, 2 mM EDTA, 1.5% w/v BSA) and then filtrated through eight layers of Miracloth. Chloroplasts and nuclei were removed by centrifugation at 1200× g for 45 s. The supernatant was further centrifuged at 5000× g for 5 min to obtain a crude mitochondrial fraction. GFP-tagged proteins were purified from crude mitochondria with the µMACS Epitope Tag Protein Isolation Kit (Miltenyi Biotec, Bergisch Gladbach, Germany) using the standard elution protocol. After separation by SDS-PAGE and transfer to the PVDF membrane, protein bands visualized by Coomassie Blue staining were excised for Edman microsequencing [43 (link)], which was performed by Proteome Factory AG (Berlin, Germany).
Macs epitope tag protein isolation kit
Manufactured by Miltenyi Biotec
Sourced in Germany
The µMACS Epitope Tag Protein Isolation Kit is a tool for purifying proteins tagged with specific epitopes. It allows for the selective isolation of target proteins from complex samples.
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4 protocols using macs epitope tag protein isolation kit
1
Isolation and Purification of Mitochondrial Proteins
2
Transfection and Immunoprecipitation of Dystroglycan
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293-Ebna cells, grown in DMEM supplemented with antibiotics and 10% (v/v) fetal calf serum, were transfected with 20 µg of wild-type or I591D DG constructs using the calcium phosphate method as described elsewhere [40] (link). After 24 h, cells were dissolved in PBS containing 1% Triton X-100 and protease inhibitors (Roche, Switzerland). Cell lysate was resolved on a 10% SDS-PAGE. For Western blot analysis, proteins were transferred to nitrocellulose and probed with an anti β-DG antibody (43-DAG) (1∶50) and with a peroxidase-conjugated secondary antibody (Sigma, USA) diluted 1∶7000 (anti-mouse); the reactive products were revealed using the luminol-based ECL system (Pierce, USA).
All the steps required for immunoprecipitation were carried out using the µMACS Epitope Tag Protein Isolation Kit (Miltenyi Biotec., Germany), following the manufacturer’s instructions. Briefly, 1 ml of total protein extract of transfected cells was incubated with 50 µl of magnetic beads conjugated with an anti-myc antibody (Miltenyi Biotec., Germany) for 30 min in ice. After several washes, the adsorbed protein was eluted with 50 µl of sample buffer and run on a 10% SDS-PAGE followed by Western blot analysis with an anti-myc antibody-HRP conjugated (Miltenyi Biotec., Germany).
All the steps required for immunoprecipitation were carried out using the µMACS Epitope Tag Protein Isolation Kit (Miltenyi Biotec., Germany), following the manufacturer’s instructions. Briefly, 1 ml of total protein extract of transfected cells was incubated with 50 µl of magnetic beads conjugated with an anti-myc antibody (Miltenyi Biotec., Germany) for 30 min in ice. After several washes, the adsorbed protein was eluted with 50 µl of sample buffer and run on a 10% SDS-PAGE followed by Western blot analysis with an anti-myc antibody-HRP conjugated (Miltenyi Biotec., Germany).
3
Immunoprecipitation of GFP and SYT1-mGFP
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We adopted a procedure described in a previous study (Tamura et al., 2013) . In brief, we prepared 1.5 g of the transgenic Arabidopsis expressing GFP or the syt1 mutant expressing ProSYT1:: SYT1-mGFP at 10 d after sowing and precipitated free GFP or SYT1-mGFP using the µMACS Epitope Tag Protein Isolation Kit (Miltenyi Biotec, Bergisch Gladbach, Germany). We checked the quality of the immunoprecipitates by flamingo staining and western blot using anti-GFP (Roche Diagnostics, Rotkreuz, Switzerland). The immunoprecipitates were briefly separated using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), and digested into peptides to identify SYT1 interactors by LC-MS/MS analyses.
4
Co-IP of YFP-D6PK and mCherry-PDK1 in Arabidopsis
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To examine the interaction between YFP-D6PK and mCherry-PDK1s in planta, crossing lines 35S::YFP-D6PK; RPS5A::mCherry-PDK1.1 and 35S::YFP-D6PK; RPS5A::mCherry-PDK1.2 were used for Co-IP assays using a µMACS Epitope Tag Protein Isolation Kit (Miltenyi Biotec), with RPS5A::mCherry-PDK1.1 and RPS5A::mCherry-PDK1.2 transgenic lines as controls. In detail, ~100 mg of 7-day-old seedlings were homogenized into 200 µL protein extraction buffer [20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.5% Triton X-100, and 1 mM DTT] containing a protease inhibitor cocktail (cOmplete, Roche) and a protein phosphatase inhibitor tablet (PhosSTOP, Roche). After centrifuge, 200 µL supernatant of each sample was transferred to a new tube for incubation with 50 µL µMACS anti-GFP magnetic microbeads, with 20 µL lysate spared as the "Input" samples. The reaction samples were incubated at 4°C for 1h, and were then loaded onto a µ column on a magnetic holder. The beads were washed 5 times with the supplied wash buffer [150 mM NaCl, 1% Igepal CA-630, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris HCl (pH 8.0)], and were then eluted with elution buffer [50 mM Tris HCl (pH 6.8), 50 mM DTT, 1% SDS, 1 mM EDTA, 0.005% bromphenol blue, 10% glycerol]. Protein samples were separated by 10% SDS-PAGE, and were eventually detected by a mouse anti-GFP HRP-conjugated antibody and a RFP antibody sequentially.
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