Western blot analysis of extracts from various cell lines, untreated or treated with peroxynitrite, degraded peroxynitrite or pervanadate, using Phospho-Tyrosine Mouse mAb (P-Tyr-100) #9411 (upper) or Nitro-Tyrosine Antibody (lower).
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To obtain optimal results with this antibody please use PVDF instead of nitrocellulose membranes.
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
Nitro-Tyrosine Antibody detects proteins and peptides containing nitro-tyrosine in a manner independent of the surrounding amino acid sequence. It is a valuable tool for identifying new nitrated proteins as well as for assaying protein nitration and measuring levels of nitrated proteins in tissues and samples. The antibody does not cross-react with unmodified tyrosine or with phospho-tyrosine. (U.S. Patent No's.: 6,441,140; 6,982,318; 7,259,022; 7,344,714; U.S.S.N. 11,484,485; and all foreign equivalents.)Species Reactivity:
Human, Mouse, Rat
Polyclonal antibodies are produced by immunizing animals with synthetic nitro-tyrosine-containing peptides . Antibodies are purified by protein A and peptide affinity chromatography.
Nitric oxide (NO) is implicated in carcinogenesis (1), chronic infection, inflammation (2) and neurodegeneration (3). High levels of both superoxide and nitric oxide in tissues interact to form peroxynitrite, a potent oxidant that can modify Tyr residues in proteins to form 3-nitro-tyrosine (4). Tyrosine nitration of mitochondrial manganese superoxide dismutase results in loss of enzymatic activity (4). The nitration of p53 at Tyr residues abolishes its capacity for binding to its DNA consensus sequence (5).
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
Use of Cell Signaling Technology (CST) Motif Antibodies within certain methods (e.g., U.S. Patents No. 7,198,896 and 7,300,753) may require a license from CST. For information regarding academic licensing terms please have your technology transfer office contact CST Legal Department at CST_ip@cellsignal.com. For information regarding commercial licensing terms please contact CST Pharma Services Department at firstname.lastname@example.org.