mtfr1 Antibody

What is MTFR1 and what are its key biological functions?

MTFR1 is a 37 kDa protein that regulates mitochondrial fission processes. It is also known as CHPPR (Chondrocyte Protein with a Poly-Proline Region) and FAM54A2. MTFR1 plays a critical role in maintaining mitochondrial organization and influences mitochondrial aerobic respiration . The protein also affects mitochondrial dynamics, linking fission processes to broader cellular functions and energy balance .

Recent studies have revealed that MTFR1 is involved in cancer progression. It promotes proliferation, invasion, migration, and enhances glycolytic capacity in lung adenocarcinoma (LUAD) cells while inhibiting apoptosis . MTFR1 has also been implicated in drug resistance mechanisms, particularly resistance to cisplatin in cancer cells .

What types of MTFR1 antibodies are commercially available and what applications are they validated for?

Multiple manufacturers offer MTFR1 antibodies with varying applications and specificities. The most commonly validated applications include:

Most commercially available MTFR1 antibodies are rabbit polyclonal antibodies, though mouse polyclonal options exist as well . Antibodies targeting different epitopes are available, including those against N-terminal, middle region, and C-terminal portions of the protein .

What species reactivity is typically observed with MTFR1 antibodies?

Most MTFR1 antibodies show reactivity to human MTFR1, with many also cross-reacting with mouse and rat orthologs due to sequence homology. According to the product specifications from multiple suppliers:

When selecting an MTFR1 antibody for cross-species applications, it's advisable to check the validation data provided by manufacturers and perform preliminary validation experiments in your specific model system.

How should I optimize Western blot protocols for MTFR1 detection?

For successful Western blot detection of MTFR1, consider the following protocol optimizations:

Sample preparation:

• NETN buffer has been successfully used for MTFR1 detection in immunoprecipitation experiments

• Include protease inhibitors in all lysis buffers

• Determine optimal protein loading (40 μg of total protein has been successful in published studies )

Electrophoresis conditions:

• 10-12% SDS-PAGE gels are recommended for optimal resolution of the 37 kDa MTFR1 protein

• Always include positive controls (A549, HCT 116, HepG2, or SW480 cells have shown good MTFR1 expression )

Antibody dilution and detection:

• Primary antibody dilutions of 1:500-1:2000 are typically effective

• Overnight incubation at 4°C may improve specific signal

• Use appropriate HRP-conjugated secondary antibodies

• MTFR1 usually appears as a single band at approximately 37 kDa

Troubleshooting:

• If high background occurs, increase blocking time or adjust antibody dilution

• For weak signals, consider longer exposure times or signal enhancement systems

• Validate results using multiple antibodies targeting different epitopes

What are the critical factors for successful immunohistochemistry with MTFR1 antibodies?

Successful immunohistochemistry (IHC) with MTFR1 antibodies requires attention to several key parameters:

Tissue fixation and processing:

• Formalin-fixed paraffin-embedded (FFPE) tissues have been successfully used with MTFR1 antibodies

• Consistent fixation times (typically 24-48 hours) are important for reproducible results

• Fresh tissue sections yield optimal results

Antigen retrieval:

• Heat-induced epitope retrieval (HIER) is generally recommended

• Test both citrate buffer (pH 6.0) and EDTA buffer (pH 9.0) to determine optimal conditions

• Ensure sufficient retrieval time while preventing tissue damage

Antibody concentration:

• Start with manufacturer-recommended dilutions (typically 1:40-1:500 for MTFR1 antibodies)

• Perform titration experiments to determine optimal concentration for your specific tissue

• Consider signal amplification systems for low-expressing tissues

Controls and interpretation:

• Include positive control tissues (thyroid cancer tissue has been documented to show strong MTFR1 expression )

• Use both positive and negative controls in every experiment

• MTFR1 typically shows cytoplasmic staining with a mitochondrial pattern

• Consider digital image analysis for quantitative assessment

How can I validate the specificity of an MTFR1 antibody for my research?

Comprehensive validation of MTFR1 antibody specificity should include multiple complementary approaches:

Genetic validation:

• Use MTFR1 knockdown/knockout controls generated with siRNA, shRNA, or CRISPR-Cas9

• Studies have successfully used shRNA constructs to knock down MTFR1 in cancer cell lines

• Observe the corresponding decrease in signal with specific antibodies

Positive and negative controls:

• Use cell lines with known MTFR1 expression levels (A549, HCT 116, HepG2, and SW480 cells express MTFR1 at detectable levels )

• Compare with non-expressing or low-expressing cell lines or tissues

Multiple detection methods:

• Confirm results across different applications (WB, IHC, IF)

• Correlate protein detection with mRNA expression data

Recombinant protein control:

• When available, use purified recombinant MTFR1 as a positive control

• Consider peptide competition assays where the immunizing peptide blocks specific binding

Multiple antibodies:

• Compare results using antibodies targeting different epitopes of MTFR1

• Consistent detection across multiple antibodies increases confidence in specificity

Expected molecular weight verification:

• Confirm detection of MTFR1 at the expected molecular weight of 37 kDa in Western blot

How can MTFR1 antibodies be used to investigate the role of MTFR1 in cancer progression?

MTFR1 antibodies are valuable tools for investigating the role of this protein in cancer progression through multiple experimental approaches:

Expression analysis in clinical samples:

• Immunohistochemistry with MTFR1 antibodies can evaluate expression across tumor stages and grades

• Studies have shown that MTFR1 is upregulated in lung adenocarcinoma tissues compared to normal lung tissues

• High MTFR1 expression correlates with poor prognosis, advanced clinical stage, lymph node metastasis, and larger tumor size in LUAD

Functional studies:

• Western blot with MTFR1 antibodies can confirm successful manipulation of MTFR1 expression in knockout or overexpression studies

• Research has demonstrated that MTFR1 overexpression stimulates proliferation, invasion, migration, and glycolytic capacity while inhibiting apoptosis of LUAD cells

• Knockdown of MTFR1 has shown opposite effects, reducing cancer cell growth and aggressiveness

Mechanistic investigations:

• Immunoprecipitation with MTFR1 antibodies can identify protein-protein interactions

• Studies have identified that MTFR1 may exert its biological functions through the AMPK/mTOR signaling pathway

• MTFR1 has also been shown to promote drug resistance via p-AKT and p-ERK/P38 signaling pathways

Regulatory pathway analysis:

• MTFR1 is directly targeted by miR-29c-3p, and this regulatory relationship can be studied using MTFR1 antibodies to confirm protein level changes

• Combined analysis of MTFR1 expression and miRNA levels can provide insights into regulatory mechanisms

Therapeutic potential assessment:

• MTFR1 antibodies can monitor changes in expression following treatment with various therapeutic agents

• Evaluating MTFR1 expression in drug-resistant cell lines (such as A549/DDP cells) can reveal its role in resistance mechanisms

How can I use MTFR1 antibodies to study mitochondrial dynamics and fission?

MTFR1 antibodies provide powerful tools for investigating mitochondrial dynamics and fission processes:

Localization studies:

• Immunofluorescence with MTFR1 antibodies can visualize the protein's distribution in relation to mitochondria

• Co-staining with mitochondrial markers (MitoTracker, TOMM20) can confirm mitochondrial localization

• Super-resolution microscopy can provide detailed analysis of MTFR1 at fission sites

Dynamic regulation analysis:

• Time-course experiments with MTFR1 antibodies can track expression changes during induced mitochondrial stress

• Western blot analysis can detect post-translational modifications that might regulate MTFR1 activity

• Subcellular fractionation followed by Western blot can assess MTFR1 distribution between cytosolic and mitochondrial compartments

Protein-protein interaction studies:

• Co-immunoprecipitation with MTFR1 antibodies can identify interactions with known fission proteins (DRP1, FIS1, MFF)

• Proximity ligation assays can visualize and quantify protein interactions in situ

• Mass spectrometry analysis of immunoprecipitated complexes can identify novel interactors

Functional perturbation approaches:

• Combined use of MTFR1 antibodies with genetic manipulation (knockdown/overexpression) can validate the effects on mitochondrial morphology

• Live-cell imaging with mitochondrial markers can track dynamic changes in mitochondrial network organization

Quantitative analysis:

• Digital image analysis of immunofluorescence data can provide metrics of mitochondrial morphology (size, number, interconnectivity)

• Correlation of MTFR1 levels with mitochondrial network parameters can establish functional relationships

What is the role of MTFR1 in drug resistance, and how can antibodies help investigate this?

MTFR1 has been implicated in cancer drug resistance, particularly to cisplatin in lung adenocarcinoma. MTFR1 antibodies enable comprehensive investigation of this phenomenon:

Expression analysis in resistant models:

• Western blot with MTFR1 antibodies can compare expression in paired sensitive and resistant cell lines

• Studies have shown that inhibition of MTFR1 expression could promote the sensitivity of A549/DDP cells (cisplatin-resistant) to cisplatin

• Immunohistochemistry can assess MTFR1 expression in patient samples before and after treatment

Signaling pathway analysis:

• MTFR1 expression has been linked to activation of p-AKT and p-ERK/P38 signaling pathways in drug-resistant cells

• Western blot with phospho-specific antibodies can monitor these pathways in relation to MTFR1 manipulation

• Co-immunoprecipitation with MTFR1 antibodies can identify relevant interacting proteins in the resistance mechanism

Experimental model design:

• Create resistance models by exposing cells to increasing concentrations of chemotherapeutic agents

• Use MTFR1 antibodies to track expression changes during resistance development

• Manipulate MTFR1 expression in sensitive and resistant cells to assess its direct role in resistance

Mechanistic investigations:

• Combine MTFR1 antibody-based detection with functional readouts like cell viability, apoptosis, and drug uptake

• Investigate metabolic alterations associated with MTFR1-mediated resistance

• Analyze mitochondrial dynamics changes in resistant cells using immunofluorescence

Therapeutic targeting assessment:

• Test combination approaches using MTFR1 inhibition with conventional chemotherapy

• Evaluate miR-29c-3p (which targets MTFR1) as a potential therapeutic approach

• Monitor MTFR1 expression changes in response to various targeted therapies

How can I address inconsistent results when using MTFR1 antibodies across different experimental platforms?

Inconsistent results across different experimental platforms when using MTFR1 antibodies can be systematically addressed through the following approaches:

Understand method-specific differences:

• Each detection method measures different aspects of MTFR1 expression and localization

• Different antibodies may recognize epitopes that are differentially accessible in various applications

• Post-translational modifications may affect antibody binding in application-specific ways

Antibody selection considerations:

• Test multiple MTFR1 antibodies targeting different epitopes

• Compare monoclonal (higher specificity) with polyclonal (multiple epitopes) antibodies

• Validate each antibody in each specific application rather than assuming cross-application performance

Protocol optimization:

• For Western blot: Optimize lysis conditions, sample preparation, and gel percentage

• For IHC/IF: Test different fixation methods, antigen retrieval protocols, and detection systems

• For IP: Evaluate different lysis buffers and binding conditions

Sample preparation variables:

• Consider the impact of cell culture conditions on MTFR1 expression

• Standardize sample collection and processing

• Test the stability of MTFR1 under various storage conditions

Quantification approaches:

• Use digital image analysis with consistent parameters

• Include calibration standards where possible

• Apply the same quantification methodology across experiments

Validation with orthogonal methods:

• Correlate protein detection with mRNA levels

• Confirm functional consequences through phenotypic assays

• Use genetic manipulation (overexpression/knockdown) to create reference standards

What are common issues encountered when using MTFR1 antibodies and how can they be resolved?

Several common issues may arise when working with MTFR1 antibodies. Here are strategies to address them:

High background in immunoblotting:

• Increase blocking time or concentration (5% BSA or milk)

• Optimize antibody concentration (test dilutions from 1:500-1:2000)

• Include additional washing steps with higher detergent concentration

• Use more specific secondary antibodies with minimal cross-reactivity

Weak or absent signal:

• Confirm MTFR1 expression in your samples (use positive controls like A549 cells )

• Try alternative extraction methods to improve protein recovery

• Increase protein loading (up to 40-50 μg)

• Consider signal enhancement systems or longer exposure times

• Test antibodies against different epitopes, as some may be inaccessible

Multiple bands in Western blot:

• Verify the expected molecular weight (37 kDa for MTFR1 )

• Include appropriate positive and negative controls

• Test freshly prepared samples to rule out degradation

• Use more specific antibodies or optimize blocking conditions

• Consider the possibility of isoforms or post-translational modifications

Poor immunohistochemical staining:

• Optimize antigen retrieval (test both citrate and EDTA-based methods)

• Adjust antibody concentration and incubation time

• Test different detection systems (polymer-based vs. ABC method)

• Consider amplification systems for low-expressing tissues

• Ensure proper tissue fixation and processing

Immunoprecipitation difficulties:

• Test different lysis buffers to maintain protein interactions

• Pre-clear lysates to reduce non-specific binding

• Increase antibody amount (0.5-4.0 μg as recommended )

• Optimize bead type and binding conditions

• Include appropriate controls (IgG, input, unbound fraction)

How should I quantify and present MTFR1 expression data for publication?

Proper quantification and presentation of MTFR1 expression data is essential for publication. Consider these best practices:

Western blot quantification:

• Use densitometry software to measure band intensity

• Normalize to appropriate loading controls (β-actin, GAPDH, or mitochondrial markers)

• Present data as fold change relative to control samples

• Include representative blot images showing all experimental conditions

• Report the molecular weight of detected bands (37 kDa for MTFR1 )

Immunohistochemistry quantification:

• Use standardized scoring systems (H-score, Allred, or percentage positive cells)

• Consider both staining intensity and proportion of positive cells

• Employ digital image analysis for objective quantification

• Have multiple observers score samples independently to ensure reliability

• Present representative images of different staining patterns

Immunofluorescence data:

• Quantify signal intensity with appropriate background subtraction

• Assess co-localization with mitochondrial markers using established metrics

• Present data as mean fluorescence intensity or percent co-localization

• Include scale bars and magnification information

• Show representative images with appropriate controls

Statistical analysis:

• Apply appropriate statistical tests based on data distribution

• Include sufficient sample sizes (n values) for statistical power

• Report both statistical significance (p-values) and effect sizes

• Use consistent statistical methods across different experiments

• Address potential confounding variables in your analysis

Data visualization:

• Present quantitative data in graphs with error bars representing variation

• Use consistent axes and scaling across related experiments

• Include all data points in addition to means when sample size is small

• Consider heat maps for complex expression patterns across multiple samples

• Use color-blind friendly palettes for accessibility

Publication requirements:

• Include detailed methods sections describing antibody source, catalog number, and dilutions

• Report specific protocols used for quantification

• Provide all necessary controls (positive, negative, loading, etc.)

• Make raw data available in supplementary materials or repositories

How can MTFR1 antibodies help investigate the relationship between mitochondrial fission and cancer metabolism?

MTFR1 antibodies provide essential tools for exploring the intricate connection between mitochondrial fission and cancer metabolism:

Expression correlation studies:

• Western blotting with MTFR1 antibodies can quantify expression across cell lines with different metabolic phenotypes

• Studies have demonstrated that MTFR1 overexpression enhances glycolytic capacity in cancer cells

• Comparing MTFR1 levels with glycolytic markers (such as GLUT1, HK2, LDHA) can establish functional relationships

Mitochondrial morphology assessment:

• Immunofluorescence with MTFR1 antibodies enables visualization of mitochondrial network fragmentation

• Research has shown that mitochondrial dynamics affect cancer cell metabolism, with fission often promoting a glycolytic phenotype

• Quantitative image analysis can correlate MTFR1 expression with mitochondrial fragmentation and metabolic parameters

Functional metabolic analysis:

• MTFR1 manipulation (knockdown/overexpression) verified by antibody detection can be correlated with:

  • Glycolytic capacity (extracellular acidification rate, glucose consumption, lactate production)

  • Oxidative phosphorylation (oxygen consumption rate, ATP production)

  • Metabolite profiles using mass spectrometry

• Studies have demonstrated that MTFR1 knockdown impairs glycolytic capacity of cancer cells

Pathway interaction studies:

• Co-immunoprecipitation with MTFR1 antibodies can identify interactions with:

  • Metabolic enzymes and regulators

  • Components of the AMPK/mTOR pathway (implicated in MTFR1 function )

  • Proteins involved in both mitochondrial dynamics and metabolism

Therapeutic targeting approaches:

• MTFR1 antibodies can monitor expression changes after treatment with:

  • Metabolic inhibitors (targeting glycolysis or OXPHOS)

  • Mitochondrial fission/fusion modulators

  • Conventional chemotherapeutics

What emerging applications of MTFR1 antibodies show the most promise for translational research?

Several emerging applications of MTFR1 antibodies show significant promise for translational research:

Predictive and prognostic biomarker development:

• MTFR1 overexpression correlates with poor prognosis in lung adenocarcinoma

• Standardized immunohistochemistry protocols with MTFR1 antibodies could enable routine assessment in clinical samples

• Combined with other markers, MTFR1 expression patterns may help stratify patients for targeted therapies

Therapeutic resistance mechanisms:

• MTFR1 has been implicated in cisplatin resistance via the p-AKT and p-ERK/P38 pathways

• Antibody-based monitoring of MTFR1 during treatment could identify resistance development

• Screening for MTFR1 expression before treatment might predict response to certain therapies

Targeted therapy development:

• As a promoter of cancer progression, MTFR1 represents a potential therapeutic target

• Antibody-based high-throughput screening could identify compounds that modulate MTFR1 expression or function

• Therapeutic modulation of the miR-29c-3p/MTFR1 axis represents another promising approach

Combination therapy strategies:

• Based on MTFR1's role in multiple signaling pathways, rational combination approaches can be developed

• MTFR1 antibodies can monitor pathway modulation during combination treatments

• Targeting both MTFR1 and its downstream effectors might overcome resistance mechanisms

Mitochondrial dynamics as a therapeutic strategy:

• Modulating mitochondrial fission through MTFR1-targeted approaches may have therapeutic potential

• MTFR1 antibodies enable verification of target engagement in preclinical models

• Monitoring mitochondrial morphology changes in response to therapy can provide mechanistic insights

Immune microenvironment interactions:

• Research has shown associations between MTFR1 expression and immune cell infiltration in cancer

• Multiplexed immunofluorescence with MTFR1 and immune markers can map these relationships

• Understanding how MTFR1-mediated metabolic changes affect the immune microenvironment could inform immunotherapy approaches