Figure 1: iNOS Protein Structure

iNOS Introduction

Protein Function

• Produces nitric oxide (NO) which is a messenger molecule with diverse functions throughout the body.
• In macrophages, NO mediates tumoricidal and bactericidal actions. Also has nitrosylase activity and mediates cysteine S-nitrosylation of cytoplasmic target proteins such COX2.
• Induced by synergistic action of endotoxins, cytokines, and IFNG/IFN-γ with bacterial lipopolysaccharides (LPS), TNF, or IL1B/interleukin-1 beta to express iNOS.

Protein Expression

• Expressed in liver, retina, bone cells and airway epithelial cells of the lung Not expressed in platelets.

Protein Localization

• Cytoplasm

Image 2: ICC Experimental Results of iNOS, Anti-iNOS [RM1017] product (ab283655).Green: iNOS, Red: alpha Tubulin, Blue: DAPI.

Isoforms & Post-Translational Modifications

• Human (P35228): Isoforms 1, 2: 126-131 kDa (predicted)
• Mouse (P29477): 131 kDa (predicted)
• Rat (Q06518): 131 kDa (predicted)
• Polyubiquitination and phosphorylation

WB Experiment Tips

Precautions

• iNOS expression is limited to certain tissues and cells, so it's crucial to include positive controls.
• Induce iNOS expression with cytokines and other factors (e.g., 0.1 µg/mL LPS treatment of RAW 264.7 cells for 6 hours).

Positive Control

• Caco-2 cell lysate
• RAW 264.7 cell lysate treated with 100 ng/ml LPS for 6 hours

Negative Control (No or weak expression)

• RAW 264.7 cell lysate
• C6 cell lysate

Example Results

Figure 3: Anti-iNOS [RM1017] (ab283655)

Primary antibody: Anti-iNOS antibody, diluted 1/1000.
Blocking: Blocked with 5% NFDM/TBST solution.

Lane 1: Caco-2 whole cell lysate.
Lane 2: RAW 264.7 whole cell lysate.
Lane 3: RAW 264.7 whole cell lysate treated with 100 ng/ml LPS for 6 hours.
Lane 4: C6 whole cell lysate.
Lane 5: C6 whole cell lysate treated with 100 ng/ml LPS for 4 hours.

Secondary antibody: Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051)at 1/100000 dilution.
Predicted band size: 131 kDa

Key control points

In the experiment, in addition to paying attention to routine issues, special attention should be paid to the following key control points:

Sample preparation:

1. Add a protease inhibitor cocktail to prevent degradation of target proteins.
2. Keep samples on ice throughout the entire sample preparation process.
3. Determine the total protein concentration of the samples using Bradford analysis, Lowry analysis, or BCA analysis.
4. Select positive and negative controls.

Electrophoresis:

1. Load at least 20μg total protein for electrophoresis.
2. It is recommended to use an 8% or lower concentration separating gel for electrophoresis.

Transfer:

1. It is recommended to stain the membrane with Ponceau S after the transfer to confirm the success of the transfer.
2. It is recommended to add SDS to the transfer buffer to a final concentration of 0.1%.
3. It is advised to use a PVDF membrane with a pore size of 0.45μm.
4. It is recommended to use 10% methanol in the transfer buffer.

References

1. Fujisawa H, Ogura T, Hokari A, Weisz A, Yamashita J, Esumi H. Inducible nitric oxide synthase in a human glioblastoma cell line. J Neurochem. 1995 Jan;64(1):85-91. doi: 10.1046/j.1471-4159.
2. Xue Q, Yan Y, Zhang R, Xiong H. Regulation of iNOS on Immune Cells and Its Role in Diseases. Int J Mol Sci. 2018 Nov 29;19(12):3805. doi: 10.3390/ijms19123805.