Anti rabbit igg hrp linked whole ab donkey

Western blotting was performed by a modification of Hara’s method and Ota’s method^{5 (link),29 (link)}. In particular, total protein extracts (2 × 10⁴ cells/lane) were separated by polyacrylamide gel electrophoresis, transferred to polyvinylidene difluoride membranes using the Trans-Blot^® Turbo Transfer system (Bio Rad Laboratories, USA), and incubated with antibodies. Immuno-labelled proteins were detected using a chemiluminescence kit (Immuno Star LD (FUJIFILM Wako Pure Chemical, Japan)) and a lumino-image analyser (ChemiDoc MP system (Bio Rad Laboratories, USA)). The primary antibodies used were Total OXPHOS Rodent WB Antibody Cocktail (abcam, UK) and rabbit anti-human HO-1 antibody (kindly provided by Dr. Shigeru Taketani)^{30 (link),31 (link)}. The secondary antibodies used were anti-rabbit IgG HRP-Linked Whole Ab Donkey (GE Healthcare, USA) for OXPHOS proteins and anti-mouse IgG HRP-Linked Whole Ab Sheep (GE Healthcare, USA) for HO-1. Beta-actin was used as an internal control. Anti-beta-actin antibody (ab8227) (abcam, UK) was used as primary antibody and anti-rabbit IgG HRP-Linked Whole Ab Donkey (GE Healthcare, USA) was used as secondary antibody.

Coimmunoprecipitation analysis was performed according to the case of AP2M^{44 (link)}. In brief, ~0.4 g of A. thaliana-expressing AP1M2-GFP, AP4M-GFP, AP2M-GFP, or free-GFP (14 DAG) were homogenized on ice in 1.2 ml of a lysis buffer (50 mM Tris-HCl, pH7.5, 1 mM EDTA, 1% Triton X-100, and a protease inhibitor cocktail in the absence of NaCl) and then centrifuged at 10,000×g for 10 min at 4 °C. The supernatants were immunoprecipitated with an anti-GFP antibody using μMACS GFP Isolation Kit (Miltenyi Biotec). Then, the immunoprecipitates were separated by SDS-PAGE and immunoblotted with an anti-GFP antibody (Clontech, No. 632375; 1:20,000) or an anti-CHC antibody (Agrisera, AS10 690; 1:2000). Anti-Mouse IgG, HRP-Linked Whole Ab Sheep (GE Healthcare, NA931; 1:5000), and Anti-Rabbit IgG, HRP-Linked Whole Ab Donkey (GE Healthcare, NA934; 1:5000) secondary antibodies were used to detect the primary antibodies with ECL select western blotting detection reagent (RPN2235, GE Healthcare). Densitometry was performed with “Quantification of Gel Bands by an Image J Macro, Band/Peak Quantification Tool⁶⁹ ”. The band intensities of CHC were divided by the band intensity of each corresponding GFP-tag, normalized to the AP1M2-GFP line, and expressed as the relative coimmunoprecipitated CHC.