tmem-135 Antibody
Description
Introduction to TMEM135 Antibody
The TMEM135 antibody is a specialized immunological reagent designed to detect and study the transmembrane protein 135 (TMEM135), also known as peroxisomal membrane protein 52 (PMP52). This antibody is critical for investigating TMEM135’s roles in mitochondrial dynamics, peroxisomal metabolism, and lipid metabolism. Below is a detailed analysis of its characteristics, applications, and research insights.
Applications in Research
TMEM135 antibodies are instrumental in diverse experimental workflows:
Western Blotting (WB)
-
Protein Quantification: Used to assess TMEM135 expression levels in mutant vs. wild-type mice, revealing structural and functional impairments in mitochondrial dynamics .
-
Post-Translational Modifications: Detected phosphorylation or ubiquitination patterns via WB, critical for studying TMEM135’s regulation of DRP1-mediated mitochondrial fission .
Immunofluorescence (IF)
-
Subcellular Localization: Demonstrated TMEM135’s colocalization with mitochondria and peroxisomes, highlighting its dual role in organelle dynamics .
-
Punctate Mitochondrial Staining: Observed in WT cells, with reduced colocalization in TMEM135 mutants, correlating with mitochondrial fusion/fission imbalances .
Immunohistochemistry (IHC)
-
Tissue-Specific Expression: Validated in human and rodent tissues, aiding in mapping TMEM135’s distribution in metabolic organs like liver and adipose tissue .
Enzyme-Linked Immunosorbent Assay (ELISA)
-
Serum or Tissue Lysate Analysis: Quantifies TMEM135 levels in clinical samples, though less commonly reported compared to WB or IF .
Mitochondrial Dynamics
-
Fission Promotion: TMEM135 antibodies identified colocalization with DRP1, a mitochondrial fission factor. Mutant TMEM135 (e.g., FUN025) disrupted DRP1 activation, leading to hyperfused mitochondria and reduced ATP production .
-
Energy Metabolism: In Tmem135 FUN025/FUN025 mice, reduced spare respiratory capacity (SRC) and maximal respiration were linked to impaired mitochondrial dynamics, underscoring TMEM135’s role in oxidative phosphorylation .
Peroxisomal Metabolism
-
β-Oxidation Regulation: TMEM135 knockdown in HepG2 cells caused triglyceride accumulation despite reduced lipogenic gene expression, implicating TMEM135 in peroxisomal β-oxidation .
-
LXR Regulation: TMEM135 is an LXR target gene; antibodies confirmed LXR agonist-induced TMEM135 upregulation in human hepatocytes and macrophages .
Cancer Biology
-
Cell Cycle Regulation: TMEM135 knockdown in HepG2 cells induced G0/G1 arrest and reduced ATP production in glucose-free media, suggesting a role in hepatocellular carcinoma progression .
Protocol Optimization
Product Specs
Constituents: 50% Glycerol, 0.01M PBS, pH 7.4
Target Background
- TMEM135 integrates biological processes related to fat metabolism and energy expenditure in both invertebrates (such as the worm) and mammalian organisms. PMID: 21151927
KEGG: cel:CELE_K02G10.3
UniGene: Cel.6106
Q&A
Basic Research Questions
How to validate TMEM135 antibody specificity in Western blotting?
-
Method: Use knockdown/knockout cell lines (e.g., siRNA-mediated TMEM135 knockdown in HepG2 cells ) alongside controls. Validate using tissues with known TMEM135 expression (e.g., human/rat liver lysates ).
-
Critical data: Compare bands at predicted molecular weights (~50–58 kDa ) and confirm absence in knockdown samples.
-
Pitfalls: Non-specific bands may arise due to protein isoforms; use antibodies targeting specific domains (e.g., C-terminal ).
What experimental models are optimal for studying TMEM135’s role in lipid metabolism?
-
In vitro: HepG2 cells for human hepatocyte studies, as TMEM135 knockdown in this line reduces β-oxidation and ATP production under glucose-free conditions .
-
In vivo: Use fasting/refeeding protocols in mice to mimic metabolic stress, but note that Tmem135 is not an LXR target in mice , limiting translational comparisons to human models.
How to resolve cross-reactivity issues in multi-species studies?
-
Approach: Validate antibody reactivity using lysates from human, mouse, and rat tissues. For example, the C-terminal antibody (ABIN6991844) detects TMEM135 in all three species , but murine LXR response elements differ from humans , necessitating functional validation.
Advanced Research Questions
How to design experiments investigating TMEM135’s dual role in peroxisomal and mitochondrial metabolism?
-
Strategy:
-
Key controls: Include assays with palmitate (peroxisomal substrate) and octanoate (mitochondrial substrate) to distinguish pathways.
How to address contradictory data on TMEM135’s role in cell proliferation?
-
Context: TMEM135 knockdown in HepG2 cells increases G0/G1 arrest and reduces ATP levels under metabolic stress , suggesting context-dependent roles.
-
Resolution:
What methodologies confirm TMEM135’s interaction with peroxisomal enzymes?
-
Proteomic workflow:
Data Integration & Interpretation
How to reconcile species-specific TMEM135 regulation in translational studies?
| Parameter | Human Models | Mouse Models |
|---|---|---|
| LXR responsiveness | Induced by LXR agonists | No significant induction |
| Metabolic role | Peroxisomal β-oxidation | Mitochondrial fission |
-
Solution: Use humanized mouse models or primary hepatocytes for studies requiring LXR-TMEM135 interplay.
What controls are essential for TMEM135 immunohistochemistry in disease models?
-
Tissues: Include positive controls (human liver ) and negative controls (TMEM135 knockdown tissues ).
-
Staining validation: Compare wild-type vs. mutant TMEM135 models (e.g., FUN025 mutation mice ) to confirm antibody specificity.
How to optimize TMEM135 detection in lipid-loaded tissues?
-
Protocol adjustments:
Methodological Pitfalls
Why do TMEM135 protein levels vary between Western blot and immunohistochemistry?
-
Key factors:
