Skip to content
Snippets Groups Projects

Compare revisions

Changes are shown as if the source revision was being merged into the target revision. Learn more about comparing revisions.

Source

Select target project
No results found

Target

Select target project
  • hhu-plant-biochemistry/Wrobel-2023-CastorBeanEndospermProteome
  • ceplas/Wrobel-2023-CastorBeanEndospermProteome
2 results
Show changes
Showing
with 13 additions and 0 deletions
![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.
\ No newline at end of file
assays/2_6-SDS-PAGE-Immunoblotting/dataset/fpls-14-1182105-g004.jpg

130 B

No preview for this file type
# 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
\ No newline at end of file