Anti-3-Nitrotyrosine antibody [7A12AF6]

• Flow Cyt

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Flow Cytometry - Anti-3-Nitrotyrosine antibody [7A12AF6] (AB110282)

HL-60 cells were stained with 1 µg/ml ab110282 following treatment with 2 mM peroxynitrite (blue) or vehicle control (red). No primary antibody control is shown in black. Peroxynitrite modifies tyrosine residues to 3-nitrotyrosine.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-3-Nitrotyrosine antibody [7A12AF6] (AB110282)

Immunocytochemistry image of ab110282 stained Human HeLa cells (A) and fibroblast cells (B, C).
Cells grown on slides were paraformaldehyde fixed (4%, 20 min) and Triton X-100 permeabilized (0.1%, 15 min). Slides were treated with/without 1 mM peroxynitrite to modify exposed tyrosines to 3-nitrotyrosine. Slides were blocked and incubated with tab110282 at 2 µg/ml overnight at 4°C. The secondary antibody (green) was Alexa Fluor® 488 Goat anti-Mouse IgG (H+L) used at a 1/1000 dilution for 1 hour. 10% Goat serum was used as the blocking agent for all blocking steps. For reference, the mitochondria (red) were identified by HSP60 / Alexa Fluor® 594 and DAPI was used to stain the cell nuclei (blue).
HeLa cells (A) and fibroblast cells (B) show surface modification of tyrosine to 3-nitrotyrosine after exposure to peroxynitrite. While (C) unexposed fibroblast cells show no modification.

• WB

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Western blot - Anti-3-Nitrotyrosine antibody [7A12AF6] (AB110282)

All lanes:

Western blot - Anti-3-Nitrotyrosine antibody [7A12AF6] (ab110282) at 1 µg/mL

Lane 1:

Bovine heart mitochondria

Lane 2:

Bovine heart mitochondria - nitrated

Lane 3:

BSA

Lane 4:

BSA - nitrated

false

• WB

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Western blot - Anti-3-Nitrotyrosine antibody [7A12AF6] (AB110282)

In this experiment, the antibody was first blocked with free nitrotyrosine before being used to blot the membrane.
The figure shows that ab110282's binding capacity was not inhibited by the free nitrotyrosine, and so only binds to the protein-bound form.

All lanes:

Western blot - Anti-3-Nitrotyrosine antibody [7A12AF6] (ab110282) at 1 µg/mL

Lane 1:

Bovine heart mitochondria

Lane 2:

Bovine heart mitochondria - nitrated

Lane 3:

BSA

Lane 4:

BSA - nitrated

false

• WB

Unknown

Western blot - Anti-3-Nitrotyrosine antibody [7A12AF6] (AB110282)

Prior to runnning the samples, the membrane was treated with sodium dithionite to reduce nitrotyrosine to aminotyrosine.

All lanes:

Western blot - Anti-3-Nitrotyrosine antibody [7A12AF6] (ab110282) at 1 µg/mL

Lane 1:

Bovine heart mitochondria

Lane 2:

Bovine heart mitochondria - nitrated

Lane 3:

BSA

Lane 4:

BSA - nitrated

false