TMEM65 Antibody

What is TMEM65 and What Are Its Key Biological Functions?

TMEM65 (Transmembrane protein 65) is primarily a mitochondrial inner-membrane protein containing three putative transmembrane regions and an N-terminal mitochondrial targeting sequence . This protein plays several critical roles in cellular physiology:

• Maintains proper cardiac intercalated disk (ICD) structure and function

• Regulates cardiac conduction velocity in the heart

• Interacts with components of the mitochondrial contact site and cristae organizing system (MICOS) complex

• Associates with SCN1B to stabilize the perinexus in cardiac ICD

• Facilitates localization of GJA1 (Connexin 43) and SCN5A to the ICD

• Regulates mitochondrial respiration and mitochondrial DNA copy number maintenance

• Recently discovered to regulate NCLX-dependent mitochondrial calcium efflux

• Promotes gastric tumorigenesis through targeting YWHAZ to activate PI3K-Akt-mTOR signaling pathway

Knockout studies in mice have demonstrated that TMEM65 is essential for survival, as Tmem65-/- mice experience growth retardation, weakness, and typically do not survive beyond postnatal day 21 .

What Applications Are TMEM65 Antibodies Validated For?

TMEM65 antibodies have been validated for multiple experimental applications:

Multiple manufacturers offer TMEM65 antibodies with different host species, clonality, and reactivity profiles. Most common are rabbit polyclonal antibodies that react with human, mouse, and rat TMEM65 .

How Can Researchers Validate the Specificity of TMEM65 Antibodies?

Validating antibody specificity is crucial for TMEM65 detection, as non-specific staining has been reported:

• Knockout/Knockdown Controls: In TMEM65 knockout heart sections, specific mitochondrial staining disappears while non-specific intercalated disc and nuclear staining may persist . siRNA-mediated silencing shows that the ~21-kDa band (endogenous TMEM65) disappears in treated samples .

• Molecular Weight Verification: Mature TMEM65 appears at ~21 kDa on Western blots, while the calculated molecular weight from amino acid sequence is ~26 kDa, indicating post-translational processing .

• Subcellular Fractionation: In proper fractionation experiments, TMEM65 predominantly appears in mitochondrial fractions .

• Band Pattern Analysis: Be aware that higher molecular weight bands (~48 kDa) observed with some anti-TMEM65 antibodies have been confirmed as non-specific .

• Multiple Antibodies Approach: Use antibodies from different sources or targeting different epitopes to confirm consistent results.

What Are the Best Protocols for Detecting TMEM65 in Mitochondrial Fractions?

For optimal detection of TMEM65 in mitochondrial preparations:

Mitochondrial Isolation:

• Use differential centrifugation followed by density gradient purification

• Confirm mitochondrial enrichment using markers like porin/VDAC1

Sample Preparation:

• Resuspend isolated mitochondria in buffer containing protease inhibitors

• Include mild detergents (0.5-1% digitonin or 1% Triton X-100) for membrane protein extraction

Western Blotting:

• Use 10-15% SDS-PAGE gels for optimal resolution of TMEM65 (20-25 kDa)

• Transfer to PVDF membranes (preferred for hydrophobic proteins)

• Block with 5% BSA or non-fat milk in TBST

• Incubate with primary anti-TMEM65 antibody at appropriate dilution (e.g., 1:1000-1:5000)

• Develop using enhanced chemiluminescence detection

Membrane Association Confirmation:

• Perform alkali extraction by treating isolated mitochondria with Na₂CO₃ (pH 11.5)

• Centrifuge to separate membrane (pellet) and soluble (supernatant) fractions

• TMEM65 should remain in the membrane fraction, confirming its integral membrane protein nature

How Do Researchers Distinguish Between Precursor and Mature Forms of TMEM65?

TMEM65 undergoes post-translational processing during mitochondrial import:

Molecular Weight Analysis:

• Full-length precursor form: ~26 kDa (calculated from amino acid sequence)

• Mature form after MTS cleavage: ~21 kDa

• Some studies identify both 19 kDa (truncated) and 25 kDa (full-length) forms

Processing Mechanism:

• N-terminal mitochondrial targeting sequence (MTS) is cleaved by mitochondrial processing peptidase (MPP)

• Cleavage site is between amino acid residues 35-64 of human TMEM65

• Potential MPP recognition site: RRL|GT between residues 52-56

Experimental Approaches:

• Compare TMEM65 bands in whole cell lysates versus purified mitochondria

• Use deletion mutants of TMEM65 fused with reporter proteins to identify processing sites

• Pulse-chase experiments can track conversion from precursor to mature form

What Controls Should Be Used When Studying TMEM65 in Knockout Models?

When working with TMEM65 knockout models, researchers should implement these controls:

Knockout Validation:

• Confirm complete loss of TMEM65 by Western blotting using validated antibodies

• Be aware that some higher molecular weight bands (~48 kDa) may persist as they represent non-specific binding

Compartment-Specific Controls:

• Use co-staining with mitochondrial markers (e.g., MitoTracker, TOMM20) to confirm loss of mitochondrial TMEM65 signal

• In cardiac tissue, distinguish between genuine intercalated disc localization and potential non-specific staining by comparing with known intercalated disc markers

Functional Controls:

• Include phenotypic assessments (e.g., growth curves, survival rates) to confirm knockout effects

• For cardiac-specific knockouts, include functional cardiac assessments to correlate molecular changes with physiological effects

Genetic Background Considerations:

• Include wild-type littermates as controls to account for genetic background effects

• Consider heterozygous animals to assess gene dosage effects

Tissue Collection Timing:

• For whole-body knockouts with lethal phenotypes, collect tissues at appropriate developmental timepoints before death (e.g., P20 for Tmem65-/- mice)

What Are the Challenges in Detecting TMEM65 in Different Subcellular Compartments?

Detecting TMEM65 across different compartments presents several technical challenges:

Antibody Specificity Issues:

• Some anti-TMEM65 antibodies show non-specific staining at intercalated discs and nuclei in heart tissue

• Higher molecular weight bands (~48 kDa) observed in Western blots with certain antibodies are non-specific

Dual Localization Interpretation:

• While primarily mitochondrial, TMEM65 has been reported at intercalated discs in cardiac tissue

• Distinguishing genuine dual localization from antibody cross-reactivity requires knockout controls

Sample Preparation Considerations:

• Different fixation methods affect epitope accessibility

• For mitochondrial proteins, maintaining mitochondrial morphology during fixation is critical

• Permeabilization conditions must balance membrane disruption for antibody access with structure preservation

Tissue-Specific Expression:

• Expression levels and potentially localization patterns vary across tissues

• In cardiac tissue specifically, mitochondrial TMEM65 staining is completely lost in knockout models, while intercalated disc staining persists, indicating non-specific binding

How Can TMEM65 Antibodies Be Used to Study Its Role in Mitochondrial Calcium Regulation?

Recent research has revealed TMEM65's role in mitochondrial calcium regulation . To investigate this function:

Co-localization Studies:

• Perform immunofluorescence co-staining with anti-TMEM65 antibodies and antibodies against calcium regulators, particularly NCLX (mitochondrial Na⁺/Ca²⁺ exchanger)

• Use super-resolution microscopy for detailed spatial analysis of protein distribution

Protein-Protein Interactions:

• Conduct co-immunoprecipitation experiments with anti-TMEM65 antibodies to identify interactions with calcium-handling proteins

• Use size exclusion chromatography with Western blotting to examine whether TMEM65 and NCLX form complexes

• Research indicates they may form a complex with molecular mass of ~140 kDa (potentially 2 NCLX:1 TMEM65)

Functional Correlation:

• In TMEM65 knockdown or overexpression models, use antibodies to confirm successful manipulation

• Then conduct calcium flux measurements using fluorescent indicators to correlate molecular changes with functional calcium handling

Pathological Models:

• Examine TMEM65 expression in conditions associated with mitochondrial calcium overload

• TMEM65 overexpression can limit cell death in response to thapsigargin or ionomycin treatment, suggesting protective effects against calcium stress

What Are the Best Approaches to Detect TMEM65 Interactions with MICOS Complex Components?

To study TMEM65 interactions with the MICOS complex:

Co-immunoprecipitation (Co-IP):

• Use anti-TMEM65 antibodies to pull down TMEM65 and probe for MICOS components (e.g., MIC60, MIC19, MIC10)

• Perform reciprocal Co-IPs using antibodies against MICOS components to confirm interactions

• Use digitonin (0.5-1%) for membrane protein complex preservation

Blue Native PAGE (BN-PAGE):

• Analyze mitochondrial complexes using BN-PAGE followed by Western blotting

• Compare complex migration patterns in control versus TMEM65-depleted samples

Proximity Ligation Assay (PLA):

• Use pairs of antibodies (anti-TMEM65 and anti-MICOS component) to perform PLA

• This generates fluorescent signals only when proteins are in close proximity (<40 nm)

Validation in Knockout Models:

• In TMEM65 knockout or knockdown models, assess changes in MICOS complex integrity

• Examine mitochondrial cristae morphology by electron microscopy in these models

How Can TMEM65 Antibodies Be Used in Cancer Research Studies?

Recent findings have implicated TMEM65 in cancer progression, particularly in gastric cancer :

Expression Analysis:

• Use anti-TMEM65 antibodies to compare expression levels between normal and cancerous tissues

• In gastric cancer, TMEM65 mRNA and protein levels are significantly upregulated compared to adjacent normal tissues

Prognostic Correlation:

• Perform immunohistochemistry on patient samples to correlate TMEM65 expression with clinical outcomes

• High TMEM65 expression predicts poor prognosis in gastric cancer patients

Mechanistic Studies:

• Use antibodies to investigate TMEM65's interaction with YWHAZ, which has been identified as a direct downstream effector

• Co-immunoprecipitation can confirm the binding of TMEM65 with YWHAZ in the cytoplasm, which inhibits ubiquitin-mediated degradation of YWHAZ

Signaling Pathway Analysis:

• After TMEM65 knockdown or overexpression, use antibodies against key signaling molecules (p-Akt, p-GSK-3β, p-mTOR) to assess pathway activation

• TMEM65 exerts oncogenic effects through activating PI3K-Akt-mTOR signaling pathway

Therapeutic Target Validation:

• Antibodies can confirm successful TMEM65 depletion in therapeutic models, such as VNP-encapsulated TMEM65-siRNA treatments that have shown significant tumor suppression in xenograft models