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Biological Description
| Specificity | Anti-IRG1 Rabbit Antibody [L17A13] detects endogenous levels of total IRG1 protein. |
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| Background | Immune-responsive gene 1 (IRG1) is among the most strongly upregulated genes in macrophages under proinflammatory conditions and is also highly expressed in the pregnant uterus during implantation. IRG1 encodes a cis-aconitate decarboxylase that catalyzes the conversion of cis-aconitic acid—an intermediate of the tricarboxylic acid (TCA) cycle—into itaconic acid. Itaconic acid acts as an endogenous inhibitor of succinate dehydrogenase, thereby linking metabolic reprogramming in macrophages to the regulation of inflammation. Functionally, IRG1 plays critical roles in embryonic implantation and neurodegeneration. In macrophages, it promotes endotoxin tolerance by enhancing A20 expression through the generation of reactive oxygen species (ROS). The cytoprotective enzyme heme oxygenase-1 (HO-1), which produces endogenous carbon monoxide (CO), is also expressed in the lung during lipopolysaccharide (LPS)-induced tolerance and cross-tolerance, highlighting a broader anti-inflammatory network in which IRG1 participates. During the early implantation phase—marked by high levels of inflammatory cytokine secretion—IRG1 expression in the uterus is particularly elevated. Dysregulation of IRG1 has been associated with autoimmune and inflammatory diseases. Localized predominantly to the mitochondria, IRG1 serves as a key link between immune and metabolic pathways. Notably, silencing IRG1 increases NF-κB and IRF3 activation, coupled with reduced A20 expression and diminished ROS production, further underscoring its regulatory role in inflammation. |
Usage Information
| Application | WB, IP | Dilution |
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| Reactivity | Human, Mouse | ||||||
| Source | Rabbit | MW | 53 kDa | ||||
| Storage Buffer | PBS, pH 7.2+50% Glycerol+0.05% BSA+0.01% NaN3 | Storage (from the date of receipt) |
-20°C (avoid freeze-thaw cycles), 2 years | ||||
| WB |
Experimental Protocol:
Sample preparation
1. Tissue: Lyse the tissue sample by adding an appropriate volume of ice-cold RIPA Lysis Buffer (containing Protease Inhibitor Cocktail)，and homogenize the tissue at a low temperature. 2. Adherent cell: Aspirate the culture medium and wash the cells with ice-cold PBS twice. Lyse the cells by adding an appropriate volume of RIPA Lysis Buffer (containing Protease Inhibitor Cocktail) and put the sample on ice for 5 min. 3. Suspension cell: Transfer the culture medium to a pre-cooled centrifuge tube. Centrifuge and aspirate the supernatant. Wash the cells with ice-cold PBS twice. Lyse the cells by adding an appropriate volume of RIPA Lysis Buffer (containing Protease Inhibitor Cocktail) and put the sample on ice for 5 min. 4. Place the lysate into a pre-cooled microcentrifuge tube. Centrifuge at 4°C for 15 min. Collect the supernatant;
5. Remove a small volume of lysate to determine the protein concentration;
6. Combine the lysate with protein loading buffer. Boil 20 µL sample under 95-100°C for 5 min. Centrifuge for 5 min after cool down on ice.
Electrophoretic separation
1. According to the concentration of extracted protein, load appropriate amount of protein sample and marker onto SDS-PAGE gels for electrophoresis. Recommended separating gel (lower gel) concentration: 10%. Reference Table for Selecting SDS-PAGE Separation Gel Concentrations 2. Power up 80V for 30 minutes. Then the power supply is adjusted (110 V~150 V), the Marker is observed, and the electrophoresis can be stopped when the indicator band of the predyed protein Marker where the protein is located is properly separated. (Note that the current should not be too large when electrophoresis, too large current (more than 150 mA) will cause the temperature to rise, affecting the result of running glue. If high currents cannot be avoided, an ice bath can be used to cool the bath.)
Transfer membrane
1. Take out the converter, soak the clip and consumables in the pre-cooled converter;
2. Activate PVDF membrane with methanol for 1 min and rinse with transfer buffer;
3. Install it in the order of "black edge of clip - sponge - filter paper - filter paper - glue -PVDF membrane - filter paper - filter paper - sponge - white edge of clip"; 4. The protein was electrotransferred to PVDF membrane. ( 0.45 µm PVDF membrane is recommended ) Reference Table for Selecting PVDF Membrane Pore Size Specifications Recommended conditions for wet transfer: 200 mA, 120 min. ( Note that the transfer conditions can be adjusted according to the protein size. For high-molecular-weight proteins, a higher current and longer transfer time are recommended. However, ensure that the transfer tank remains at a low temperature to prevent gel melting.)
Block
1. After electrotransfer, wash the film with TBST at room temperature for 5 minutes;
2. Incubate the film in the blocking solution for 1 hour at room temperature;
3. Wash the film with TBST for 3 times, 5 minutes each time.
Antibody incubation
1. Use 5% skim milk powder to prepare the primary antibody working liquid (recommended dilution ratio for primary antibody 1:1000), gently shake and incubate with the film at 4°C overnight; 2. Wash the film with TBST 3 times, 5 minutes each time;
3. Add the secondary antibody to the blocking solution and incubate with the film gently at room temperature for 1 hour;
4. After incubation, wash the film with TBST 3 times for 5 minutes each time.
Antibody staining
1. Add the prepared ECL luminescent substrate (or select other color developing substrate according to the second antibody) and mix evenly;
2. Incubate with the film for 1 minute, remove excess substrate (keep the film moist), wrap with plastic film, and expose in the imaging system. (Exposure time of at least 90s is recommended)
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References
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