Purified PorK/N complexes were resuspended in 10 mM Tris-HCl, pH 7.5, 50 mM NaCl, 10% glycerol and 0.5% DDM. Native Page G-250 sample additive (Life Technologies) was added to the resuspended sample to a final concentration of 0.125% before loading a NativePAGE 3–12% Bis-Tris gel. The gel was subjected to electrophoresis at 150 V for 1 hour at 4°C then the voltage was increased to 250 V until the dye front reached the bottom of the gel. Supercomplexes, pyruvate dehydrogenase complex of ≥ 10000 kDa and the tetrametric form of ATP synthase of 2400 kDa [32 (link)] were obtained from bovine heart mitochondria (Abcam), solubilized in 1% digitonin and used as molecular weight standards.
Bovine heart mitochondria
Manufactured by Abcam
Bovine heart mitochondria are isolated organelles obtained from the heart tissue of bovine (cattle) origin. Mitochondria are the cellular organelles responsible for the production of energy in the form of ATP through the process of oxidative phosphorylation. The bovine heart mitochondria product provides a reliable source of these essential cellular components for research purposes.
Automatically generated - may contain errors
Lab products found in correlation
Nativepage g 250 sample additive, by Thermo Fisher Scientific (1 mentions)
Western blotting detection reagent, by GE Healthcare (1 mentions)
Anti mouse igg horseradish peroxidase hrp, by GE Healthcare (1 mentions)
Bn page, by Thermo Fisher Scientific (1 mentions)
Imagequant las 4000, by GE Healthcare (1 mentions)
2 protocols using bovine heart mitochondria
1
Native PAGE Analysis of Mitochondrial Complexes
2
Fractionation and Immunodetection of Mitochondrial Complexes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The solubilized mitochondria and the bovine heart mitochondria (Abcam) with the surfactant were fractionated into each complex using the native page TM bis-tris gel system (BN-PAGE ; Invitrogen, California, United State) with 3!12% bis-tris protein gels, 1.0 mm (Invitrogen). Protein was transferred from the electrophoresis gel onto a PVDF (polyvinylidene fluoride) membrane by electroblotting. Each protein was immunodetected using specific antibodies. An ImageQuant LAS 4000 (GE Healthcare Japan, Tokyo, Japan) was used for measurement, and the value of each sample was calculated as a ratio relative to that of the bovine heart mitochondria. The western blotting detection reagents were purchased from GE Healthcare.
The following primary antibodies were purchased from Abcam : complex I, anti -NDUFA9 (20C11B11B11) ; complex II, anti -SDHA (2E3GC12FB2AE2) ; complex III, anti-UQCRC2 (13G12AF12BB 11) ; complex IV, anti -COX IV (20E8C12) ; complex V, anti -ATP5A (15H4C4). The secondary antibody used was anti mouse IgGhorse radish peroxidase (HRP) (GE Healthcare).
The following primary antibodies were purchased from Abcam : complex I, anti -NDUFA9 (20C11B11B11) ; complex II, anti -SDHA (2E3GC12FB2AE2) ; complex III, anti-UQCRC2 (13G12AF12BB 11) ; complex IV, anti -COX IV (20E8C12) ; complex V, anti -ATP5A (15H4C4). The secondary antibody used was anti mouse IgGhorse radish peroxidase (HRP) (GE Healthcare).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!