MTCH2 Antibody

What is MTCH2 and what are its primary functions in cellular biology?

MTCH2 (mitochondrial carrier homolog 2) is a protein primarily localized to the mitochondrial outer membrane where it functions as a protein insertase that mediates insertion of transmembrane proteins. Specifically, MTCH2 catalyzes insertion of proteins with alpha-helical transmembrane regions, such as signal-anchored, tail-anchored, and multi-pass membrane proteins, but does not mediate insertion of beta-barrel transmembrane proteins . The protein has a calculated molecular weight of 33 kDa (303 amino acids) .

MTCH2 serves multiple critical cellular functions including:

• Acting as a receptor for pro-apoptotic BH3-interacting domain death agonist (p15 BID), playing a critical role in apoptosis regulation

• Regulating quiescence and cycling of hematopoietic stem cells (HSCs)

• Functioning as a regulator of mitochondrial fusion, essential for naive-to-primed interconversion of embryonic stem cells

• Contributing to lipid homeostasis and adipocyte differentiation

• Supporting mitochondrial function and energy metabolism in various cell types

Research has demonstrated that MTCH2 dysregulation is implicated in several cancer types, including castration-resistant prostate cancer and ovarian cancer, highlighting its importance as a potential biomarker and therapeutic target .

What are the validated applications for MTCH2 antibodies in research?

MTCH2 antibodies have been validated for multiple research applications, each offering distinct advantages for specific experimental questions. Based on technical validation data, MTCH2 antibodies can be reliably used in the following applications:

It is important to note that optimal antibody concentrations should be determined for each experimental system through careful titration to achieve the best signal-to-noise ratio .

What species reactivity is confirmed for commercially available MTCH2 antibodies?

MTCH2 antibodies have been tested and validated in multiple species, with varying degrees of confirmed reactivity. When selecting an antibody for your research, it is essential to consider species compatibility:

For species not explicitly confirmed, researchers should consider protein homology between species. Many antibody manufacturers indicate that strong sequence homology between species may predict cross-reactivity, but experimental validation is always recommended before proceeding with extensive studies .

How is MTCH2 expression correlated with cancer progression and clinical parameters?

Recent bioinformatic analyses have revealed significant associations between MTCH2 expression and clinical parameters in various cancer types. In prostate cancer, MTCH2 overexpression is associated with critical clinical parameters, indicating its potential role as a prognostic marker . Single-cell sequencing data demonstrates elevated MTCH2 expression specifically in the prostate cancer epithelium .

In castration-resistant prostate cancer (CRPC):

• MTCH2 is upregulated in locally treated CRPC tissue

• Higher expression is observed in various primary human CRPC cells

• MTCH2 expression correlates with disease progression and poor clinical outcomes

Research using genetic manipulation approaches (shRNA and CRISPR-mediated knockout) has demonstrated that MTCH2 depletion significantly impairs mitochondrial function in CRPC cells, resulting in:

• Reduced oxygen consumption rate

• Diminished complex I activity

• Decreased ATP levels

• Mitochondrial depolarization

• Increased reactive oxygen species production

These metabolic alterations led to inhibited cell viability, proliferation, and migration, with increased apoptosis in primary CRPC cells. Conversely, ectopic expression of MTCH2 enhanced ATP production and promoted cell proliferation and migration .

In ovarian cancer, MTCH2 has been shown to play roles in energy metabolism and metastatic potential. Experimental manipulation of MTCH2 expression levels demonstrably affects cell migration and invasion capabilities, with overexpression enhancing and knockdown reducing these cancer-associated behaviors .

What controls should be included when validating MTCH2 antibody specificity?

Proper validation of MTCH2 antibody specificity is crucial for experimental reliability. Based on published research methodologies, the following controls should be included:

• Genetic manipulation controls:

  • MTCH2 knockdown via siRNA (transient) or shRNA (stable)

  • MTCH2 knockout via CRISPR-sgRNA approaches

  • MTCH2 overexpression using expression plasmids

• Detection controls:

  • Positive control lysates from cells known to express MTCH2 (e.g., HEK-293, HeLa, HepG2)

  • Negative control using secondary antibody only

  • Isotype control using irrelevant antibody of same isotype and concentration

  • Blocking peptide competition assay when available

• Technique-specific controls:

  • For Western blotting: Size marker to confirm expected 33 kDa band

  • For IHC/IF: Include both positive (known expressing) and negative tissues

  • For IP/Co-IP: Include IgG control

Published studies have successfully used MTCH2 siRNA to reduce protein expression, confirming antibody specificity when the signal decreases proportionally to the knockdown efficiency . Similarly, CRISPR-sgRNA approaches provide definitive validation of antibody specificity when the signal is absent in knockout cell lines .

How can researchers investigate MTCH2 protein interactions with other mitochondrial proteins?

MTCH2 interacts with multiple proteins as part of its various cellular functions. Research has demonstrated several approaches to investigate these interactions:

• Co-immunoprecipitation (Co-IP):

  • Successfully used to identify interactions between MTCH2 and other proteins

  • Published studies have documented interactions between MTCH2, claudin-3, and AIMP2 (aminoacyl tRNA synthetase-interacting multifunctional protein 2)

  • Recommended protocol: Use 0.5-4.0 μg of MTCH2 antibody for 1.0-3.0 mg of total protein lysate

• Proximity ligation assays:

  • Allows visualization of protein interactions in situ

  • Can detect endogenous protein interactions without overexpression

• Functional interaction studies:

  • Investigate how knockdown of one protein affects the others

  • Example: MTCH2 knockdown led to downregulation of AIMP2 expression levels, while AIMP2 knockout did not affect MTCH2 expression but significantly decreased claudin-3 expression

Research has revealed that MTCH2 interacts with apoptotic factors like p15 BID, suggesting a role in apoptotic signaling pathways . Additional interaction partners include proteins involved in mitochondrial fusion and lipid homeostasis, consistent with MTCH2's multiple cellular functions .

What considerations should be made when studying MTCH2 in different cellular compartments?

MTCH2 is primarily localized to the mitochondrial outer membrane, but studying its distribution and function requires careful consideration of subcellular fractionation and localization techniques:

• Subcellular fractionation:

  • Mitochondrial isolation procedures should be optimized to maintain outer membrane integrity

  • Differential centrifugation with gradient purification is recommended

  • Western blot analysis should include compartment-specific markers:

    • Outer mitochondrial membrane: TOM20, VDAC

    • Inner mitochondrial membrane: Complex IV subunits

    • Matrix: HSP60

    • Cytosolic: GAPDH or β-actin

• Immunofluorescence microscopy:

  • Co-staining with mitochondrial markers (MitoTracker or TOM20) is essential

  • Fixation method can significantly impact results:

    • 4% paraformaldehyde preserves structure while maintaining antigenicity

    • Cold methanol fixation may be preferred for membrane proteins

  • Recommended dilution for IF/ICC applications: 1:50-1:500

• Functional assays for mitochondrial compartments:

  • Outer membrane permeabilization: Cytochrome c release assays

  • Respiration studies: Oxygen consumption measurements

  • Membrane potential: JC-1 or TMRE staining

  • ATP production: Luminescence-based assays

Research has shown that MTCH2 depletion impacts mitochondrial function across multiple parameters, including oxygen consumption rate, complex I activity, ATP levels, membrane potential, and reactive oxygen species production .

What are the optimal protocols for detecting MTCH2 by Western blotting?

Western blotting is one of the most common applications for MTCH2 antibodies. The following protocol has been validated for optimal results:

• Sample preparation:

  • Use RIPA buffer with protease inhibitors for cell lysis

  • For mitochondrial enrichment, consider differential centrifugation

  • Load 20-40 μg of total protein per lane

  • Include positive control lysates (HEK-293, HeLa, or HepG2 cells)

• Gel electrophoresis and transfer:

  • 10-12% SDS-PAGE is suitable for resolving the 33 kDa MTCH2 protein

  • Transfer to PVDF membrane is preferred over nitrocellulose

  • Use wet transfer method for better efficiency with membrane proteins

• Antibody incubation:

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

  • Primary antibody dilution: 1:2000-1:14000 in blocking buffer

  • Incubate overnight at 4°C for optimal results

  • Secondary antibody: HRP-conjugated anti-rabbit IgG at 1:5000-1:10000

• Detection and analysis:

  • Enhanced chemiluminescence (ECL) detection

  • Expected band: 33 kDa (observed molecular weight)

  • For quantification, normalize to appropriate loading controls (β-actin, GAPDH, or mitochondrial markers like VDAC)

This protocol has been successfully used in multiple published studies investigating MTCH2 expression in various cell types and tissues .

What are the recommended protocols for immunohistochemical detection of MTCH2?

Immunohistochemistry (IHC) allows visualization of MTCH2 expression in tissue samples. The following protocol is recommended based on validated methods:

• Sample preparation:

  • Fix tissues in 10% neutral buffered formalin

  • Embed in paraffin and section at 4-5 μm thickness

  • Mount sections on positively charged slides

• Antigen retrieval:

  • Primary recommendation: TE buffer pH 9.0

  • Alternative method: Citrate buffer pH 6.0

  • Heat-induced epitope retrieval using pressure cooker or microwave

• Staining procedure:

  • Block endogenous peroxidase: 3% H₂O₂ in methanol

  • Block non-specific binding: 5% normal goat serum

  • Primary antibody: 1:50-1:500 dilution

  • Incubate overnight at 4°C or 1-2 hours at room temperature

  • Detection: HRP-polymer detection system with DAB chromogen

  • Counterstain: Hematoxylin

• Controls and analysis:

  • Positive control: Human liver tissue has been validated

  • Negative control: Omit primary antibody

  • Analysis: Assess staining pattern (membranous/mitochondrial), intensity, and distribution

  • Consider digital image analysis for quantification

IHC studies have successfully detected MTCH2 in various tissues including human liver , prostate cancer samples , and ovarian cancer specimens , with primarily mitochondrial and membranous staining patterns.

How should researchers optimize co-immunoprecipitation experiments with MTCH2 antibodies?

Co-immunoprecipitation (Co-IP) is valuable for studying MTCH2 protein interactions. The following protocol has been validated for MTCH2 Co-IP experiments:

• Cell lysis and preparation:

  • Use mild lysis buffer (e.g., 25 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% NP-40, 1 mM EDTA, 5% glycerol)

  • Add protease and phosphatase inhibitors freshly

  • Clear lysate by centrifugation (14,000 × g, 10 min, 4°C)

  • Pre-clear lysate with protein A/G beads to reduce non-specific binding

• Immunoprecipitation:

  • Use 0.5-4.0 μg MTCH2 antibody per 1.0-3.0 mg of total protein lysate

  • Incubate with lysate overnight at 4°C with gentle rotation

  • Add protein A/G beads and incubate for 1-2 hours

  • Wash beads 3-5 times with lysis buffer

• Analysis:

  • Elute bound proteins with SDS sample buffer

  • Analyze by SDS-PAGE and Western blotting

  • Probe for potential interacting proteins (e.g., claudin-3, AIMP2)

  • Include IgG control and input sample on all blots

• Validation approaches:

  • Reverse Co-IP: Immunoprecipitate with antibody against suspected interacting protein

  • Cross-linking prior to lysis can capture transient interactions

  • Proximity ligation assay as independent confirmation

Successful Co-IP experiments have demonstrated interactions between MTCH2 and both claudin-3 and AIMP2, revealing a tripartite interaction among these proteins that may regulate mitochondrial function .

What analytical methods should be used to quantify MTCH2 expression levels?

Accurate quantification of MTCH2 expression is critical for comparative studies. Several validated approaches include:

• Western blot densitometry:

  • Capture images within linear dynamic range

  • Use software like ImageJ, Image Lab, or similar

  • Normalize to appropriate loading controls

  • For mitochondrial proteins, consider normalizing to:

    • Total protein (Ponceau S or REVERT stain)

    • Mitochondrial markers (VDAC, TOM20)

  • Present data as fold change relative to control

• qRT-PCR for transcript analysis:

  • Design primers specific to MTCH2 transcript

  • Use reference genes appropriate for tissue/treatment

  • Calculate relative expression using 2^(-ΔΔCt) method

  • Validate protein changes with Western blot

• Immunohistochemistry quantification:

  • Digital pathology approaches using color deconvolution

  • Score staining intensity (0-3+) and percentage of positive cells

  • Calculate H-score or Quick score

  • Use automated image analysis software when possible

• Flow cytometry (for cell studies):

  • Requires cell permeabilization for intracellular staining

  • Use fluorochrome-conjugated secondary antibodies

  • Include isotype control

  • Present as mean fluorescence intensity (MFI)

These methods have been successfully employed to demonstrate differential MTCH2 expression in normal versus cancer tissues, and to quantify changes following genetic manipulation .

What are common challenges when working with MTCH2 antibodies and how can they be addressed?

Researchers may encounter several technical challenges when working with MTCH2 antibodies. Here are evidence-based solutions to common issues:

• Low or no signal in Western blot:

  • Increase protein loading (40-60 μg total protein)

  • Optimize antibody concentration (try higher concentration within 1:50-1:500 range)

  • Extend primary antibody incubation (overnight at 4°C)

  • Try alternative antigen retrieval methods for fixed samples

  • Check sample preparation (include protease inhibitors)

  • Consider more sensitive detection methods (ECL Plus or Femto)

• Multiple bands or non-specific binding:

  • Increase blocking stringency (5% BSA instead of milk)

  • Add 0.1-0.3% Triton X-100 to reduce hydrophobic interactions

  • Increase washing duration and number of washes

  • Validate specificity with knockdown/knockout controls

  • Use fresh antibody aliquots (avoid freeze-thaw cycles)

• High background in immunofluorescence:

  • Optimize fixation protocol (try 4% PFA vs. methanol)

  • Extend blocking time (2 hours at room temperature)

  • Use Image-iT FX Signal Enhancer before antibody incubation

  • Include 0.1% Tween-20 in antibody dilution buffer

  • Prepare fresh mounting medium with anti-fade reagent

• Poor immunoprecipitation efficiency:

  • Increase antibody amount (up to 4.0 μg per sample)

  • Try different lysis buffers to maintain protein interactions

  • Cross-link antibody to beads to prevent co-elution

  • Extend incubation time for antigen-antibody binding

Researchers have successfully addressed these challenges by optimizing antibody concentrations and incubation conditions, as evidenced by published studies using MTCH2 antibodies in various applications .

How can researchers validate knockdown or knockout models when studying MTCH2 function?

Proper validation of MTCH2 genetic manipulation models is essential for functional studies. The following approaches have been successfully used in published research:

• siRNA/shRNA knockdown validation:

  • Western blot confirmation of protein reduction

  • Multiple siRNA sequences targeting different regions

  • Include scrambled/non-targeting control

  • Quantify knockdown efficiency (typically 70-90% reduction)

  • Check for off-target effects on related proteins

  • Monitor expression over time for transient knockdown

• CRISPR-Cas9 knockout validation:

  • Western blot confirmation of complete protein absence

  • PCR and sequencing of targeted genomic region

  • Multiple sgRNA designs to control for off-target effects

  • Single-cell cloning and screening

  • Functional validation of mitochondrial phenotypes

• Overexpression model validation:

  • Western blot confirmation of increased protein levels

  • Include empty vector control

  • Verify subcellular localization using fractionation or IF

  • Monitor potential toxicity from overexpression

• Functional validation:

  • Mitochondrial function assays (oxygen consumption, ATP production)

  • Apoptosis measurements

  • Cell proliferation and migration assays

  • Phenotype rescue experiments

Published studies have demonstrated that genetic silencing via shRNA and knockout through CRISPR-sgRNA approaches effectively deplete MTCH2, leading to impaired mitochondrial function and altered cellular behaviors . Similarly, ectopic expression of MTCH2 enhances ATP production and promotes cancer cell proliferation and migration .