TMEM68 Antibody
Description
TMEM68 Antibody Overview
TMEM68 antibodies target the human transmembrane protein 68, a 324-amino-acid polypeptide with a molecular weight of 37.4 kDa . The protein is anchored to the ER membrane via two transmembrane domains (TMDs), with both its N- and C-termini exposed to the cytosol . Antibodies are available in polyclonal formats, primarily raised in rabbits, and detect epitopes such as residues 74–114 of the human protein .
Key Applications
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Immunodetection: ELISA, Western blot (WB), and immunofluorescence (IF) .
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Tissue Localization: Highest TMEM68 mRNA and protein levels are observed in the brain .
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Functional Studies: Investigating TMEM68's roles in lipid metabolism and lipid droplet formation .
3.1. TMEM68's Role in Lipid Metabolism
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Acyltransferase Activity: TMEM68 exhibits monoacylglycerol acyltransferase (MGAT) and diacylglycerol acyltransferase (DGAT) activities, critical for triacylglycerol (TG) synthesis .
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Lipid Droplet Formation: Overexpression of TMEM68 increases TG levels by 1.5–6.1-fold in neuroblastoma (SK-N-SH) and glioblastoma (U251) cells, promoting lipid droplet expansion .
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Metabolic Regulation: TMEM68 upregulates lipogenic genes (e.g., DGAT1, FASN) and modifies glycerophospholipid composition, including phosphatidylcholine and phosphatidylethanolamine .
3.3. Disease Relevance
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Cancer Models: TMEM68 knockdown reduces TG storage by 25–31% in glioblastoma cells, linking it to lipid-driven oncogenesis .
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Brain-Specific Expression: Elevated TMEM68 levels in the brain suggest specialized roles in neuronal lipid homeostasis .
Validation and Challenges
Product Specs
**Constituents:** 50% Glycerol, 0.01M PBS, pH 7.4
Target Background
Q&A
How can researchers validate the specificity of TMEM68 antibodies in Western blotting?
Validation of TMEM68 antibodies requires a multi-step approach combining immunoblotting, knockout controls, and epitope tagging. In studies using COS-7 cells expressing His₆- or FLAG-tagged TMEM68, antibodies against these tags detected bands at ~42 kDa (His₆-TMEM68) and ~39 kDa (TMEM68-FLAG), consistent with predicted molecular weights . Critical controls include:
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Knockout validation: Compare lysates from wild-type and TMEM68-knockout cells (e.g., SK/TMEM68 KO2) to confirm signal absence in null backgrounds .
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Membrane fractionation: Demonstrate antibody reactivity exclusively in membrane pellets (100,000 × g), as TMEM68 is an integral ER protein insoluble in sodium carbonate buffers .
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Epitope competition: Pre-incubate antibodies with excess immunogenic peptides (e.g., residues 51–75 for TMD1-specific antibodies) to block binding .
Table 1: TMEM68 Antibody Validation Checklist
What are the recommended controls for TMEM68 antibody-based subcellular localization studies?
ER localization of TMEM68 requires stringent verification due to its atypical targeting mechanism independent of classical ER retention signals . Essential controls include:
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Co-staining with ER markers: Use antibodies against calnexin or protein disulfide isomerase (PDI) alongside TMEM68. In COS-7 cells, TMEM68-GFP shows >90% co-localization with ER-tracker dyes .
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Protease protection assays: Treat membrane vesicles with proteinase K ± Triton X-100. Cytosolic-facing termini (His₆/FLAG tags) should be degraded regardless of detergent, while luminal markers like PDI require detergent for proteolysis .
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TMD deletion mutants: Express ΔTMD1-GFP constructs; loss of ER localization confirms TMD1’s role in membrane targeting .
Which tissue types show the highest TMEM68 expression levels?
Quantitative PCR analysis of murine tissues revealed brain as the predominant site of TMEM68 expression, with 3–5× higher mRNA levels than in liver or adipose tissue . This aligns with TMEM68’s proposed role in neural lipid metabolism.
Table 2: TMEM68 mRNA Expression Across Tissues (ΔΔCt Method)
| Tissue | Relative Expression (vs. 36B4) | Biological Replicates |
|---|---|---|
| Brain | 8.7 ± 0.9 | n=3 |
| Liver | 2.1 ± 0.3 | n=3 |
| Adipose | 1.8 ± 0.2 | n=3 |
How can researchers resolve discrepancies in TMEM68 localization data between studies?
Conflicting reports on TMEM68’s association with lipid droplets (LDs) versus ER membranes often stem from:
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Antibody cross-reactivity: Commercial antibodies may recognize unrelated LD proteins. Validate using TMEM68-KO cells and confirm ER localization via co-staining with Sec61β .
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Overexpression artifacts: TMEM68 overexpression (e.g., SK/TMEM68 cells) increases TAG synthesis, indirectly enlarging LDs . Use endogenous expression models and titrate antibody concentrations to avoid false-positive LD signals.
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Fixation conditions: Methanol fixation better preserves ER structures than paraformaldehyde for immunofluorescence .
What methodological considerations are critical when quantifying TMEM68 in lipid metabolism studies?
TMEM68’s role in TAG synthesis requires integration of antibody-based protein quantification with lipidomic assays:
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Parallel lipid extraction: After immunoblotting, use the same lysates for thin-layer chromatography (TLC) to measure TAG, DAG, and MAG levels .
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Normalization strategy: Express TMEM68 protein levels relative to ER markers (e.g., calnexin) rather than total protein, as TMEM68 abundance correlates with ER content .
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Functional blocking: Use TMEM68 antibodies conjugated to agarose beads for immunodepletion experiments; assess TAG synthesis rates in depleted lysates .
How can researchers optimize TMEM68 antibody protocols for co-localization studies with ER markers?
Achieving precise co-localization (>90% overlap) demands:
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Dual tagging: Express TMEM68-FLAG alongside ER-targeted mCherry-Sec61β. Perform sequential immunostaining to avoid fluorophore cross-talk .
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Super-resolution imaging: Use stimulated emission depletion (STED) microscopy to distinguish ER tubules (20–50 nm diameter) from potential background signals .
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Quantitative analysis: Calculate Manders’ overlap coefficients (MOC) using ImageJ plugins, with thresholds set via TMEM68-KO negative controls .
