assays/2_6-SDS-PAGE-Immunoblotting/protocols/SDS-PAGE-Immunoblotting.md

+# 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