ABN249
Anti-HNE Michael Adducts Antibody, Reduced
serum, from rabbit
Sinonimo/i:
Reduced 4-hydroxy-trans-2-nonenal Michael adducts, Reduced HNE Michael adducts
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About This Item
Codice UNSPSC:
12352203
eCl@ss:
32160702
NACRES:
NA.41
Prodotti consigliati
Origine biologica
rabbit
Livello qualitativo
Forma dell’anticorpo
serum
Tipo di anticorpo
primary antibodies
Clone
polyclonal
Reattività contro le specie
bovine, human
tecniche
western blot: suitable
Condizioni di spedizione
ambient
modifica post-traduzionali bersaglio
unmodified
Descrizione generale
Reactive aldehydes generated during lipid peroxidation contribute to cell and tissue damage associated with oxidative stress. Alpha,beta-unsaturated carbonyl compounds such as 4-hydroxy-2-nonenal (HNE), 4-oxo-2-nonenal (ONE), and acrolein, generated from oxidation of polyunsaturated fatty acids (PUFAs), readily interact with nucleophilic groups on proteins, leading to intramolecular or intermolecular protein crosslinking. Two types of HNE modifications exist, the first type is formed by a 1:1 Michael adduct reaction with lysine amine, histine imidazole, or cysteine sulfhydryl, while the second type is produced by one HNE reacting simultaneously with two free amine groups from two N-alpha-acetyllysines (NALs), so-called Michael-Schiff base adduct. In the latter case, the initial nonfluorescent 2:1 lysine-HNE Michael adduct-Schiff base further undergoes oxidative cyclization to form a fluorescent 2-hydroxy-3-imino-1,2-dihydropyrrol derivative commonly referred to as HNE fluorophore. Reversibly formed HNE-Lys Michael adduct is not stable and reduction by sodium borohydride (NaBH4) is often employed to preserve Lys HNE mofidication in protein samples. Reductive stabilization by NaBH4 treatment is also used to prevent 4-HNE loss from 4-HNE-Cys Michael adduct by retro-Michael addition reaction (RMA) during collision-induced dissociation (CID) and electron transfer dissociation (ETD) MS/MS.
Specificità
Target modification is not species-specific.
This polyclonal antiserum detected HNE-modified BSA only after, but not before, reduction by sodium borohydride.
Immunogeno
HNE-modifed KLH chemically reduced by sodium borohydride.
Applicazioni
Anti-HNE Michael Adducts Antibody, Reduced, Cat. No. ABN249, is a highly specific rabbit polyclonal antibody that targets the reduced form of 4-Hydroxy-2-noneal Michael adducts and has been tested in Western Blotting.
Application Notes:
1. ABN249 replaces Calbiochem 393207. ABN249 is expected to work in immunocytochemistry and immunohistochemistry. Please refer to the following 393207 citations as examples of sample preparations and detection in these two types of applications:
Usatyuk, P.V., et al. (2006). J. Biol. Chem. 281(46):35554-35566 (Immunocytochemistry).
McCormack, A.L., et al. (2005). J. Neurochem. 93(4):1030-1037 (Immunohistochemistry).
2. Anti-HNE Fluorophore, Cat. No. 393206, is also available for the detection of HNE Fluorophore by ELISA, Immunohistochemistry, and Western blotting.
3. To generate a positive control for Western blotting, treat a 2 mg/mL BSA solution in 10 mM KH2PO4, pH 7.4, with 0.1 to 0.5 mM HNE and incubate for 1 hr at 37°C. The reaction can be monitored by the decrease of HNE absorbance at 224 nm (extinction coefficient 13,750/M). Free HNE can be removed by gel filtration and dialysis following the chemical reaction.
HNE Michael adducts in protein solutions must be chemically reduced by sodium borohydride (NaBH4) prior to analysis using ABN1348. A freshly prepared 100 mM NaBH4 solution in 1 mM NaOH is added 1:9 (V:V) to the protein sample solution to a final NaBH4 concentration of 10 mM. The protein solution is kept at room temperature for 10 to 30 min prior to quenching with the addition of 5 to 10 molar equivalents of acetone.
1. ABN249 replaces Calbiochem 393207. ABN249 is expected to work in immunocytochemistry and immunohistochemistry. Please refer to the following 393207 citations as examples of sample preparations and detection in these two types of applications:
Usatyuk, P.V., et al. (2006). J. Biol. Chem. 281(46):35554-35566 (Immunocytochemistry).
McCormack, A.L., et al. (2005). J. Neurochem. 93(4):1030-1037 (Immunohistochemistry).
2. Anti-HNE Fluorophore, Cat. No. 393206, is also available for the detection of HNE Fluorophore by ELISA, Immunohistochemistry, and Western blotting.
3. To generate a positive control for Western blotting, treat a 2 mg/mL BSA solution in 10 mM KH2PO4, pH 7.4, with 0.1 to 0.5 mM HNE and incubate for 1 hr at 37°C. The reaction can be monitored by the decrease of HNE absorbance at 224 nm (extinction coefficient 13,750/M). Free HNE can be removed by gel filtration and dialysis following the chemical reaction.
HNE Michael adducts in protein solutions must be chemically reduced by sodium borohydride (NaBH4) prior to analysis using ABN1348. A freshly prepared 100 mM NaBH4 solution in 1 mM NaOH is added 1:9 (V:V) to the protein sample solution to a final NaBH4 concentration of 10 mM. The protein solution is kept at room temperature for 10 to 30 min prior to quenching with the addition of 5 to 10 molar equivalents of acetone.
Qualità
Evaluated by Western Blotting in Reduced HNE-BSA.
Western Blotting Analysis: A 1:10,000 of this antibody detected 10 ng of HNE-modified BSA only after, but not before, reduction by sodium borohydride.
Western Blotting Analysis: A 1:10,000 of this antibody detected 10 ng of HNE-modified BSA only after, but not before, reduction by sodium borohydride.
Descrizione del bersaglio
Variable depending on the size(s) of the HNE-modified protein(s). Uncharacterized bands may be observed in some lysate(s).
Linkage
Replaces: Calbiochem 393207
Risultati analitici
Control
HNE-modifed BSA chemically reduced by sodium borohydride.
HNE-modifed BSA chemically reduced by sodium borohydride.
Altre note
Concentration: Please refer to lot specific datasheet.
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Codice della classe di stoccaggio
12 - Non Combustible Liquids
Classe di pericolosità dell'acqua (WGK)
WGK 1
Punto d’infiammabilità (°F)
Not applicable
Punto d’infiammabilità (°C)
Not applicable
Certificati d'analisi (COA)
Cerca il Certificati d'analisi (COA) digitando il numero di lotto/batch corrispondente. I numeri di lotto o di batch sono stampati sull'etichetta dei prodotti dopo la parola ‘Lotto’ o ‘Batch’.
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