Analysis Note

ControlHNE-modifed BSA chemically reduced by sodium borohydride.

Application

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.

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.

General description

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.

Immunogen

HNE-modifed KLH chemically reduced by sodium borohydride.

Linkage

Replaces: Calbiochem 393207

Other Notes

Concentration: Please refer to lot specific datasheet.

Quality

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.

Specificity

Target modification is not species-specific.

This polyclonal antiserum detected HNE-modified BSA only after, but not before, reduction by sodium borohydride.

Target description

Variable depending on the size(s) of the HNE-modified protein(s). Uncharacterized bands may be observed in some lysate(s).