Fluorescein labeling of proteins located near NO NitO-Stamp™ , NO-Responsive Protein Labeling Reagent

NitO-Stamp™ is a nitric oxide (NO)-responsive protein labeling reagent that are activated in the presence of a high concentration of cellular NO and labels proteins with fluorescein. By adding the reagent and fixing the cells, NO localization can be visualized under a fluorescence microscopy. NitO-Stamp™ is also useful for identifying and analyzing proteins proximal to NO.

NitO-Stamp™ Product Image

Nitric oxide (NO) is a small gaseous molecule with a molecular weight of 30 and plays diverse roles in biological systems.
Below are some areas where the physiological significance of NO is actively studied:

Physiologically, NO is produced from L-arginine by NO synthases (NOS) and acts on biomolecules such as proteins. One of the actions of NO is to regulate protein functions via the formation of nitrosylation and nitration of proteins. Because NO is unstable in a cellular environment and has a short half-life, it is thought that NO acts in a limited local area. Therefore, monitoring NO-rich areas in cells and analyzing proteins located in proximity to NO are critical issues to understand its physiological roles.

NitO-Stamp™ consists of an NO-reactive moiety and a fluorescein moiety linked by a spacer. It remains inactive in the absence of NO but becomes activated upon reaction with NO. Once activated, it rapidly reacts with nucleophilic groups on nearby proteins, labeling them with fluorescein.

Conventional NO-responsive fluorescent probes become fluorescent upon reacting with NO. While they can visualize NO generation in live cells, they diffuse throughout the cell after reaction, making them unsuitable for observing NO localization. They also cannot be used in fixed cells due to washout during fixation. On the other hand, NitO-Stamp™ can visualize the NO-rich regions after cell fixation whereas it cannot detect NO in live cells prior to fixation due to excessive fluorescence from unreacted NitO-Stamp™ .

※ Refer to examples below.

RAW264.7 cells (murine macrophage cell line) were immunostimulated (treated with 0.5 μg/mL LPS, 7.5 ng/mL INF-γ) in the presence or absence of iNOS inhibitor 1400W (400 μM) and incubated for 20 hours. After adding 10 μM NitO-Stamp™, the cells were cultured for 60 minutes. Confocal laser scanning microscope imaging was performed after cell fixation with chilled methanol. Immunostaining of the fixed cells was performed using anti-calnexin (rabbit) as 1st antibody and anti-Rb IgG-Alx633 as 2nd antibody.

10 μM NitO-Stamp™ was added to RAW264.7 cells in which immune stimulation (treated with 0.5 μg/mL LPS, 7.5 ng/mL INF-γ for 20 hours) was given (+) or not (-), and the cells were cultured for 60 min. After preparing the cell lysates, immunoprecipitation was performed with an anti-fluorescein antibody to enrich fluorescein-labeled proteins. Proteins obtained by immunoprecipitation were applied to in-gel trypsin digestion to produce peptide solutions. The peptide solutions (+) and (-) were treated with TMT heavy tag and TMT light tag, respectively, the two samples were mixed in a 1:1 ratio and subjected to LC-MS/MS analysis to investigate changes between samples comprehensively. Figures show the experimental scheme of proteomics analysis (left) and volcano plot of protein signal changes by immune stimulation (+)/(-) (right).

Nishikawa, Y., et al., "Development of a Nitric Oxide-Responsive Labeling Reagent for Proteome Analysis of Live Cells.", ACS Chem. Biol., 14(3), 397–404 (2019). [PMID：30715847]