IDH3 Antibody
Description
Subunit-Specific Detection
IDH3 antibodies target distinct subunits:
-
IDH3A: Catalytic subunit essential for isocitrate decarboxylation to α-ketoglutarate (α-KG) .
-
IDH3B: Regulates substrate binding and allosteric regulation by ADP/NADH .
-
IDH3G: Modulates TCA cycle activity via redox-sensitive interactions .
Antibody Types and Applications
Notes:
-
WB: Western blotting.
-
IHC-P: Immunohistochemistry (paraffin-embedded).
-
ICC/IF: Immunocytochemistry/Immunofluorescence.
-
IP: Immunoprecipitation.
Cancer Biology
-
Glioblastoma (GBM): IDH3A overexpression correlates with reduced TCA cycle flux and increased glycolysis, promoting tumor growth. Antibodies confirmed elevated IDH3A in GBM patient samples .
-
Esophageal Squamous Cell Carcinoma (ESCC): IDH3B overexpression is linked to poor survival (5-year OS: 40.7% vs. 57.6% in negative cases). IHC studies demonstrated higher IDH3B levels in metastatic lymph nodes .
Neurodegenerative Diseases
-
Alzheimer’s Disease (AD): IDH3β knockdown in mice exacerbated tau phosphorylation and synaptic loss. Overexpression restored α-KG levels and reduced AD-like pathology .
-
Retinal Degeneration: Mutations in IDH3A and IDH3B cause retinal degeneration. Antibodies identified reduced IDH3 activity in mutant mice .
Metabolic Regulation
-
One-Carbon Metabolism: IDH3A interacts with cytosolic serine hydroxymethyltransferase (cSHMT), enhancing thymidylate synthesis. Antibody-based studies revealed extramitochondrial IDH3A localization during S-phase .
IDH3B in ESCC
Data derived from ESCC patient cohorts .
IDH3A in Psychiatric Disorders
Lower cerebellar IDH3A expression is observed in bipolar disorder, major depressive disorder, and schizophrenia, suggesting mitochondrial dysfunction as a contributing factor .
Experimental Challenges and Solutions
-
Antibody Specificity: Polyclonal antibodies (e.g., ab228596) may cross-react with homologous subunits. Pre-adsorption controls and knockout models are recommended .
-
Localization Studies: Mitochondrial vs. cytosolic IDH3A detection requires ΔMTS mutants (lacking mitochondrial targeting signals) to confirm extramitochondrial functions .
Emerging Trends
-
Redox Regulation: IDH3γ undergoes reversible redox modifications (e.g., disulfide bonding) that modulate TCA cycle activity. Antibodies identified H₂O₂-sensitive IDH3γ in cardiomyocytes .
-
Therapeutic Targeting: IDH3A inhibition reduces α-KG production, limiting HIF-1α stabilization and angiogenesis in cancer models .
Product Specs
**Constituents:** 50% Glycerol, 0.01M PBS, pH 7.4
Target Background
- Four IDH genes were expressed across all plant organs examined. One gene (At4g35650) exhibited predominant expression in pollen. In leaves, IDH genes displayed high expression levels in the veins and, to a lesser extent, in mesophyll cells. PMID: 16527867
Q&A
Basic Research Questions
How to validate IDH3 antibody specificity in mammalian tissue models?
-
Perform Western blot against recombinant IDH3 protein expressed in E. coli BL21/pET28a-idh3 systems to confirm target band alignment at ~40 kDa .
-
Include siRNA-mediated IDH3 knockdown controls in cell lines (e.g., HepG2) to verify signal reduction .
-
Compare cross-reactivity profiles against IDH3α, IDH3β, and IDH3γ subunits using isoform-specific knockout models .
What experimental applications are suitable for IDH3 antibodies in metabolic studies?
How to optimize IDH3 antibody protocols for liver cancer models?
-
Use AFB1-induced hepatocarcinogenesis models to correlate IDH3 expression trends (mRNA vs. protein) via qPCR and immunohistochemistry .
-
Include time-course experiments (e.g., 0–72 hr AFB1 exposure) to track dynamic changes .
Advanced Research Questions
How to resolve contradictory data between IDH3 mRNA and protein expression levels?
-
Conduct pulse-chase assays to measure protein half-life under stress conditions (e.g., AFB1) .
-
Assess post-transcriptional regulators (e.g., microRNAs) via RNA-seq and proteomic profiling .
-
Example: In 5xFAD Alzheimer’s models, IDH3β protein decreases despite stable mRNA, suggesting translational suppression .
What strategies mitigate cross-reactivity in IDH3 subunit-specific studies?
-
Design epitope-specific antibodies using bioinformatic tools (e.g., hydrophobicity plots; Figure 4 in ).
-
Validate with in silico docking simulations against IDH3α/β/γ tertiary structures .
-
Combine immunoprecipitation (IP) with mass spectrometry to identify off-target binding partners .
How to optimize co-immunoprecipitation (Co-IP) for IDH3 interactome studies?
-
Use crosslinkers (e.g., DSP) to stabilize weak protein interactions .
-
Screen buffer conditions (e.g., 150 mM NaCl, 1% Triton X-100) to reduce non-specific binding .
-
Validate interactions with reciprocal IP and in situ proximity ligation assays .
What controls are critical for IDH3 functional studies in neurodegenerative disease models?
-
Include age-matched wild-type and 5xFAD transgenic mice to benchmark IDH3β depletion effects on synaptic markers (PSD95, synaptophysin) .
-
Pair immunohistochemistry with enzymatic activity assays (NAD+/NADH ratios) to link expression to metabolic dysfunction .
Methodological Troubleshooting
How to address non-specific bands in IDH3 Western blots?
Why do IDH3 antibody signals vary between fresh-frozen and paraffin-embedded tissues?
-
Prolonged formalin fixation masks epitopes; optimize antigen retrieval using high-pH Tris-EDTA .
-
Compare signal intensity in parallel-processed samples to quantify fixation-induced antigen loss .
