MGST3 Antibody

What is MGST3 and what are its primary biological functions?

MGST3 plays a dual role in cellular processes with both glutathione S-transferase and glutathione peroxidase activities. It primarily facilitates the conversion of lipophilic substances into more water-soluble products, thus assisting in cellular detoxification processes . Additionally, MGST3 catalyzes the conjugate addition of reduced glutathione to the alpha, beta-unsaturated C=C carbonyl group of eicosanoids such as leukotriene A4 and 15-deoxy-Delta12,14-prostaglandin J2. These reactions form GSH adducts that are relevant to the inflammatory response . The enzyme also catalyzes glutathione-dependent reduction of eicosanoid peroxides to yield corresponding eicosanoid hydroxides .

MGST3 displays high expression levels in liver and kidney tissues, indicating its significant role in these major detoxification centers . Recent research has also identified its involvement in Alzheimer's disease pathophysiology through regulation of BACE1 protein translation and subsequent amyloidogenesis .

What types of MGST3 antibodies are available for research applications?

Based on current research tools, two main types of MGST3 antibodies are prominently available:

When selecting an antibody for your research, consider your experimental application needs. The mouse monoclonal antibody has been successfully used in immunoprecipitation protocols to pull down the 15kDa MGST3 protein from human and rat liver samples, with smaller yields from HepG2 lysate and mouse liver lysate . The rabbit recombinant monoclonal antibody has been validated for Western blot applications with A673 and HepG2 cell lysates .

How should I optimize Western blot conditions for MGST3 detection?

For optimal Western blot detection of MGST3:

• Sample preparation: Use fresh tissue samples with particular attention to liver and kidney tissues where MGST3 is highly expressed . For cell lines, HepG2 cells have shown good MGST3 expression levels .

• Antibody dilution: The rabbit recombinant monoclonal antibody (ab192254) has been successfully used at 1/10000 dilution for Western blot applications . Start with the manufacturer-recommended dilution and optimize if necessary.

• Expected band size: The predicted molecular weight of MGST3 is approximately 17 kDa . Verify that your detected band matches this expected size.

• Controls: Include positive controls such as HepG2 cell lysate when establishing your protocol .

• Detection method: A secondary antibody such as Goat Anti-Rabbit IgG (H+L) has been successfully used with the rabbit monoclonal antibody .

How can MGST3 antibodies be utilized in Alzheimer's disease research?

Recent studies have revealed that MGST3 plays a significant role in Alzheimer's disease (AD) pathophysiology through regulation of BACE1 protein translation and amyloidogenesis . When investigating MGST3 in AD research:

• Expression analysis: MGST3 protein levels are significantly increased in the hippocampus of APP/PS1 mice (an AD model) compared to wild-type mice. Similar overexpression is observed in SY5Y-APP cells compared to SH-SY5Y cells . Use validated MGST3 antibodies to quantify this upregulation in your experimental models.

• Knockdown studies: MGST3 knockdown has been shown to reduce BACE1 protein levels and subsequent amyloidogenesis. This approach provides a valuable tool for understanding the mechanistic relationship between MGST3 and AD pathology .

• Pathway analysis: Investigate the relationship between MGST3, RGS4, and AKT signaling. MGST3 knockdown reduces phosphorylated AKT levels, which affects BACE1 translation. MGST3 antibodies can be used in combination with antibodies against these pathway components to track signaling changes .

• Dual immunofluorescence: Co-staining with antibodies against MGST3 and BACE1 can help visualize their cellular co-localization and relationship in various experimental conditions .

The proposed model suggests that enhanced MGST3 protein in the hippocampus of AD promotes RGS4 expression, which enhances BACE1 translation via p-AKT signaling, leading to enhanced APP processing and Aβ generation .

What are the best methodological approaches for immunofluorescence detection of MGST3?

For optimal immunofluorescence detection of MGST3:

• Cell preparation: Seed cells in confocal dishes and allow them to achieve appropriate confluence (typically 60-70%) .

• Fixation protocol: Wash cells three times with PBS, then fix with 4% paraformaldehyde at 37°C for 30 minutes. After washing, permeabilize with 0.3% Triton X-100 at room temperature for 10 minutes .

• Blocking: Block with 10% donkey serum at 37°C for 30 minutes to minimize non-specific binding .

• Antibody dilution: Use MGST3 antibody at 1:100 dilution (for PA5-97969 from Thermo Fisher Scientific) . Incubate at 4°C for 18 hours for optimal staining.

• Secondary antibody: Use appropriate secondary antibodies such as IFKine green donkey anti-goat IgG (1:200) or IFKine red donkey anti-rabbit IgG (1:200), depending on your primary antibody host species .

• Nuclear counterstain: Use 4′,6-diamidino-2-phenylindole (DAPI) for nuclear counterstaining .

• Imaging: Utilize confocal microscopy (e.g., Leica TCS SP8 X) for high-resolution imaging of MGST3 cellular localization .

How can MGST3 knockdown experiments be designed and validated?

When designing MGST3 knockdown experiments:

• siRNA design: Select validated siRNA sequences targeting MGST3. Multiple siRNAs should be tested to confirm specificity .

• Transfection optimization: Optimize transfection conditions in your specific cell line. For SH-SY5Y cells, 48-hour transfection has been effective .

• Knockdown validation: Confirm MGST3 knockdown by:

  • Western blot: Use validated MGST3 antibodies to quantify protein reduction

  • qRT-PCR: Measure MGST3 mRNA levels to confirm transcript reduction

  • Immunofluorescence: Visualize decreased cellular MGST3 levels

• Controls: Include appropriate controls:

  • Non-targeting siRNA as negative control

  • Housekeeping protein detection (e.g., GAPDH) for normalization

• Downstream analysis: After confirming knockdown, proceed with:

  • Analysis of target proteins (e.g., BACE1, APP, RGS4)

  • Functional assays relevant to your research question

  • Pathway analysis (e.g., AKT signaling)

How does MGST3 function in oxidative stress response?

MGST3 has glutathione transferase and glutathione peroxidase activities that contribute to cellular protection against oxidative stress . When investigating MGST3's role in oxidative stress:

• Oxidative stress models: H₂O₂ treatment (e.g., 800μM) of cultured cells can serve as an in vitro oxidative stress model . MGST3 antibodies can be used to track changes in expression under these conditions.

• ROS measurement: Combine MGST3 detection with ROS measurement assays to correlate MGST3 levels with cellular oxidative state. Interestingly, research has shown that MGST3 knockdown does not necessarily change ROS levels despite affecting apoptotic markers .

• Pathway analysis: Investigations have found that several ROS-related genes are differentially altered by MGST3 knockdown, including GGT5 (downregulated), and NQ01, OSGIN1, and GDF15 (upregulated) .

• Apoptosis markers: MGST3 knockdown has been shown to reduce the protein level of caspase12, suggesting involvement of ER stress pathways . MGST3 antibodies can be used alongside apoptotic marker antibodies to investigate these relationships.

What is the relationship between MGST3 and protein-protein interactions in neurodegenerative disease models?

Recent research has identified MGST3's role in regulating protein interactions relevant to neurodegenerative diseases:

• UBL3-α-synuclein interaction: Silencing MGST3 significantly downregulates the interaction between UBL3 and α-synuclein . This finding is particularly relevant to Parkinson's disease research, where α-synuclein aggregation is a key pathological feature.

• Experimental approach: Split-luciferase complementation assays can be used to study this interaction, where cells are co-transfected with NGluc-UBL3 and α-syn-CGluc constructs . MGST3 antibodies can be used to confirm knockdown efficiency in these experimental setups.

• Oxidative stress effects: The interaction between UBL3 and α-synuclein can be further modulated by oxidative stress, which is relevant given MGST3's role in oxidative stress response .

• Combined knockdown studies: When designing experiments to study these interactions, researchers should consider the combined effects of MGST3 knockdown and oxidative stress induction, as this may provide insights into disease mechanisms .

What are the optimal immunoprecipitation protocols for MGST3?

For effective immunoprecipitation of MGST3:

• Antibody selection: Both mouse monoclonal (2G10CG7, ab110309) and rabbit recombinant monoclonal (EPR12352, ab192254) antibodies have been validated for immunoprecipitation applications .

• Sample source: Human and rat liver samples have yielded good results with the mouse monoclonal antibody, while HepG2 lysate and mouse liver lysate yielded smaller amounts .

• Validation approach: Consider using mass spectrometry to confirm the identity of the immunoprecipitated protein, as has been done with the mouse monoclonal antibody pulling down the 15kDa MGST3 protein .

• Expected results: When using immunoprecipitation followed by SDS-PAGE and Coomassie staining, expect to see a band at approximately 17 kDa, which is the predicted size of MGST3 .

How can MGST3 antibodies be used in multiparameter experimental designs?

For complex experimental designs involving MGST3:

• Co-immunoprecipitation: Use MGST3 antibodies to identify interaction partners by co-immunoprecipitation followed by mass spectrometry or Western blot analysis.

• Dual immunofluorescence: MGST3 antibodies can be used alongside antibodies against functionally related proteins (e.g., BACE1, RGS4) for co-localization studies . For example, when studying MGST3's role in Alzheimer's disease, dual staining with MGST3 and BACE1 antibodies can reveal their spatial relationship.

• Pathway analysis: Combine MGST3 antibodies with antibodies against signaling proteins like phosphorylated AKT to track pathway activities in response to experimental manipulations .

• Animal models: In animal studies such as those using APP/PS1 mice, MGST3 antibodies can be used to track expression changes in disease models compared to controls .

What are common challenges in Western blot detection of MGST3?

When troubleshooting Western blot issues with MGST3 detection:

• Signal specificity: Ensure the detected band is at the expected molecular weight of approximately 17 kDa . Multiple bands may indicate non-specific binding or protein degradation.

• Sample preparation: MGST3 is expressed at high levels in liver and kidney tissues . If using other tissue types or cell lines, protein levels may be lower, requiring loading more protein or using more sensitive detection methods.

• Antibody dilution: While 1/10000 dilution has been successful with the rabbit recombinant monoclonal antibody , optimization may be necessary for your specific experimental conditions.

• Detection method: If signal is weak, consider using enhanced chemiluminescence (ECL) substrates with longer exposure times or more sensitive detection systems.

• Membrane type: PVDF membranes may give better results than nitrocellulose for some antibodies due to higher protein binding capacity.

How can the specificity of MGST3 antibodies be validated in experimental systems?

To validate MGST3 antibody specificity:

• Knockdown controls: Use MGST3 siRNA to create negative control samples where MGST3 expression is reduced . The antibody signal should correspondingly decrease in these samples.

• Overexpression controls: Complementary to knockdown, overexpression of MGST3 should result in increased antibody signal.

• Peptide competition: Pre-incubation of the antibody with the immunizing peptide should eliminate specific binding.

• Cross-species reactivity: If your research involves multiple species, verify that the antibody recognizes MGST3 in all relevant species. Both highlighted antibodies react with human, mouse, and rat samples .

• Alternative antibodies: Where possible, validate findings using multiple antibodies targeting different epitopes of MGST3.

• Mass spectrometry: For definitive validation, immunoprecipitated proteins can be analyzed by mass spectrometry to confirm identity, as demonstrated with the mouse monoclonal antibody .