ATP5A1 Monoclonal Antibody (7H10BD4F9)

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	-Mouse (1:500)

Protocol tips
-Mouse (1:500)

Publication protocol

Briefly, isolated large cerebral arteries and microvessels were homogenized in ice-cold NP40 lysis buffer (Invitrogen, Frederick, MD) supplemented with proteinase inhibitor cocktail (Cat No. P8340; Sigma Aldrich, St. Louis, MO) and phosphatase inhibitor cocktail (Cat No. P2850; Sigma Aldrich, St. Louis, MO) and then centrifuged and the supernatant was analyzed. Pierce BCA protein assay (Thermo Scientific, Rockford, IL) was used to determine protein concentration. We then separated protein samples using gel electrophoresis on a 4–20% SDS-PAGE gradient gel. Proteins were transferred onto a polyvinylidene difluoride membrane, blocked with casein blocking buffer (#92740200, Li-cor, Lincoln, NE), washed, and incubated overnight with primary antibodies in casein blocking buffer at 4°C. The following primary antibodies for mitochondrial and non-mitochondrial proteins were used: Anti-Complex II Fp subunit I at 1:1000 dilution (70 KDa; #459200, Invitrogen, Frederick, MD); Anti-Complex III Subunit I core at 1:1000 dilution (53 KDa; #459140, Invitrogen, Frederick, MD); ATP synthase Complex V subunit alpha at 1:500 dilution (50 KDa; #459240, Invitrogen, Frederick, MD) etc. After incubation with the primary antibody, membranes were washed and incubated at room temperature for 90 min in 1%BSA-TBST with secondary goat anti-rabbit IgG at 1:2500 dilution (#7074S, Cell Signaling Technology, Danvers, MA) or goat anti-mouse IgG at 1:5000 dilution (#7076P2, Cell Signaling Technology, Danvers, MA). Immunobands were visualized using chemiluminescence (LumiGLO, Gaithersburg, MD) and autoradiography. Then, immunobands were scanned and analyzed using ImageJ software. We quantified the optical density of each band and normalized it to the intensity of the corresponding β-actin band.

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