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  • hhu-plant-biochemistry/Wrobel-2023-CastorBeanEndospermProteome
  • ceplas/Wrobel-2023-CastorBeanEndospermProteome
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# 2.5 Isolation of organellar membranes
Membranes were isolated from the collected organellar fractions (F1-4) as described by Fujiki et al. (1982) and Reumann et al. (1995) with modifications. The obtained fractions were slowly diluted with hypo-osmotic buffer (20 mM HEPES-KOH, pH 6.8 and 0.8 mM MgCl2) and incubated on ice for 30 minutes to osmotically disrupt the organelles. The suspension was subjected to 10 freeze/thaw cycles (freezing in liquid nitrogen, thawing at room temperature) to lyse efficiently the organelles. After each thaw cycle the sample was homogenized by mixing. Membranes were sedimented by centrifugation at 100,000 g at 4°C for 1 h and washed in 100 mM sodium carbonate (pH 11.5) to remove membrane- associated proteins. A second centrifugation step (100,000 g at 4°C for 1 h) was performed to harvest the organellar membranes. The membrane pellet was resuspended in 20 mM HEPES-KOH, pH 6.8 and 0.8 mM MgCl2 and stored at -80°C for further experiments. Concentration of the membrane proteins were determined using Pierce BCA protein assay kit (ThermoFisher Scientific).
- Fujiki, Y., Hubbard, A. L., Fowler, S., and Lazarow, P. B. (1982). Isolation of intracellular membranes by means of sodium carbonate treatment: application to endoplasmic reticulum. J. Cell Biol. 93, 97–102. doi: 10.1083/jcb.93.1.97
- Reumann, S., Maier, E., Benz, R., and Heldt, H. W. (1995). The membrane of leaf peroxisomes contains a porin-like channel. J. Biol. Chem. 270, 17559–17565. doi: 10.1074/jbc.270.29.17559
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![fpls-14-1182105-g004.jpg](dataset\fpls-14-1182105-g004.jpg)
**Figure 4**
Purification of intracellular membranes from castor bean endosperm. Distribution of organellar membrane proteins within the organellar fractions (F1-F4) obtained after sucrose density gradient centrifugation. (A) SDS-PAGE analysis. Each lane was loaded with enriched membrane proteins from each pellet fraction after organelle disruption and ultracentrifugation (F1/F2: 25 µg, F3/F4: 15 µg). Equal loading of the protein gel within the fractions was monitored by staining with colloidal Coomassie G-250 dye. (B) Immunoblot analysis using antibodies raised against the following organelle membrane proteins: chloroplast TIC40, mitochondrial AOX, ER BIP2, and peroxisomal PEX14 proteins. Expected molecular weight for the corresponding castor bean proteins: 50.1 kDa for RcTIC40 (A3QSJ7), 39-41 kDa for RcAOX1/2 (B9RXE2/B9SO38), 73.3 kDa for RcBIP2 (B9RYP6), and 58.3 kDa for RcPEX14 (B9RB25). The membrane was serially probed with antibodies to the indicated marker proteins. The positions of molecular mass markers (in kDa) are indicated at the left.
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assays/2_6-SDS-PAGE-Immunoblotting/dataset/fpls-14-1182105-g004.jpg

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# 2.6 SDS-PAGE and immunoblotting
Membrane proteins from each organellar fraction (F1-4) were separated on an SDS-polyacrylamide gel and visualized using Colloidal Coomassie staining G-250 dye (Laemmli, 1970). To determine the purity of the organellar fractions via immunoblotting, membrane proteins separated by SDS-PAGE were transferred to a 0.2 μm polyvinylidene difluoride membrane, which was probed with primary antibodies against the following organellar membrane marker proteins: rabbit a-AOX (Agrisera, 1:1,000), rabbit a-BIP2 (Agrisera, 1:5,000), rabbit a-TIC40 (1:2,500), and rabbit a-PEX14 (Agrisera, 1:12,500). A horseradish peroxidase-conjugated goat a-rabbit IgG secondary antibody (Merck, 1:2,500) was used for chemiluminescence detection. The membrane was serially probed with antibodies to the indicated proteins. Imaging was performed with ImageQuant LAS 4000 Mini Bimolecular Imager (GE Healthcare) using exposure times between 1/8 and 15 seconds.
- Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685. doi: 10.1038/227680a0
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