Western blot analysis of extracts from various cell lines using MT-ND3 Antibody (upper) and GAPDH (D16H11) XP® Rabbit mAb #5174 (lower).
Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA and 50% glycerol. Store at –20°C. Do not aliquot the antibody.
For western blots, incubate membrane with diluted primary antibody in 5% w/v BSA, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight.
NOTE: Please refer to primary antibody datasheet or product webpage for recommended antibody dilution.
From sample preparation to detection, the reagents you need for your Western Blot are now in one convenient kit: #12957 Western Blotting Application Solutions Kit
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
Load 20 µl onto SDS-PAGE gel (10 cm x 10 cm).
NOTE: Volumes are for 10 cm x 10 cm (100 cm2) of membrane; for different sized membranes, adjust volumes accordingly.
* Avoid repeated exposure to skin.
posted June 2005
revised November 2013
Reprobing of an existing membrane is a convenient means to immunoblot for multiple proteins independently when only a limited amount of sample is available. It should be noted that for the best possible results a fresh blot is always recommended. Reprobing can be a valuable method but with each reprobing of a blot there is potential for increased background signal. Additionally, it is recommended that you verify the removal of the first antibody complex prior to reprobing so that signal attributed to binding of the new antibody is not leftover signal from the first immunoblotting experiment. This can be done by re-exposing the blot to ECL reagents and making sure there is no signal prior to adding the next primary antibody.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalently purified water.
posted June 2005
revised October 2016
Protocol Id: 10
MT-ND3 Antibody recognizes endogenous levels of total MT-ND3 protein.Species Reactivity:
Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues near the carboxy terminus of human MT-ND3 protein. Antibodies are purified by peptide affinity chromatography.
Mitochondrially encoded NADH dehydrogenase 3 (MT-ND3) is encoded by the mitochondrial gene MT-ND3 and is a core subunit of the mitochondrial membrane respiratory chain NADH dehydrogenase (Complex I). Located in the mitochondrial inner membrane, Complex I is the largest of the five respiratory complexes and catalyzes electron transfer from NADH to ubiquinone. The mitochondrially encoded subunits of Complex I, including MT-ND3, are the most hydrophobic subunits and form the core of the transmembrane region (1,2). Pathogenic variants of the MT-ND3 gene are known to cause mitochondrial complex I deficiency (MT-C1D) and may lead to a wide range of clinical disorders, including Leigh syndrome, Leber hereditary optic neuropathy, and encephalopathy (3-5).
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