MTERF1 Antibody

What is MTERF1 and what cellular functions does it control?

MTERF1 facilitates the control of gene expression within the mitochondria, influencing the synthesis of essential components for the electron transport chain . As a transcription termination factor, it binds to a 28 bp region within the tRNA(Leu(uur)) gene at a position immediately adjacent to and downstream of the 16S rRNA gene . This binding promotes DNA bending and partial unwinding, enabling MTERF1 to prevent antisense transcription over ribosomal RNA genes . The protein contains three leucine zippers that form a three-stranded coiled-coil structure that binds to DNA .

Methodologically, researchers investigating MTERF1 function should consider its dual roles in:

• Transcription termination (with polar directionality)

• Replication regulation through contrahelicase activity

What experimental techniques are MTERF1 antibodies validated for?

MTERF1 antibodies have been validated for multiple experimental applications with specific methodological considerations:

For optimal results, researchers should validate antibody specificity using positive controls (U-2 OS cells, RT4 cells) and negative controls (MTERF1 knockdown samples) .

How should researchers differentiate between MTERF family members when selecting antibodies?

When designing experiments to differentiate MTERF family members:

• Select antibodies targeting unique epitopes - MTERF1 antibodies raised against amino acids 1-200 of the 399-amino acid protein minimize cross-reactivity with other family members (MTERFD1, MTERFD2, MTERFD3)

• Validate specificity through:

  • Western blot analysis confirming the expected molecular weight (~39 kDa for MTERF1)

  • Immunoprecipitation followed by mass spectrometry

  • Peptide competition assays using the immunizing peptide

• Consider functional distinctions:

  • MTERF1: Primarily acts as transcription termination factor

  • MTERFD1: Functions as mitochondrial transcription regulator with two splice isoforms

  • MTERFD3: Involved in cell cycle regulation and cell growth

How can researchers investigate MTERF1's directional contrahelicase activity using antibodies?

MTERF1 exhibits a unique directional contrahelicase activity that blocks mtDNA unwinding by the mitochondrial helicase TWINKLE . To study this mechanism:

• Design rolling-circle templates with MTERF1 binding sites in different orientations:

  • Forward orientation (replication progressing in same direction as rRNA transcription initiated at HSP)

  • Reverse orientation (replication progressing opposite to rRNA transcription)

• Implement ChIP approaches:

  • Cross-link protein-DNA complexes using 1% formaldehyde (10 min at room temperature)

  • Quench with glycine (125 mM final concentration)

  • Isolate mitochondria and perform MTERF1 immunoprecipitation

• Analyze protein-DNA interactions:

  • Use validated MTERF1 antibodies (typically at 4 μg/ml concentration) for immunoprecipitation

  • Perform qPCR or sequencing on precipitated DNA

  • Compare binding patterns at different mtDNA regions

• Functional validation:

  • Reconstitute replication systems with purified components (POLγ, mtSSB, TWINKLE)

  • Add recombinant MTERF1 and monitor effects on DNA synthesis using radiolabeled dNTPs

  • Analyze replication products on denaturing gels

This approach reveals MTERF1's direction-dependent effect on mtDNA replication - strongly inhibiting replication when the MTERF1 site is in the reverse orientation but having minimal effect in the forward orientation .

What protocols maximize detection sensitivity for MTERF1 in tissues with low expression levels?

When investigating tissues with low MTERF1 expression, researchers should employ these methodological optimizations:

• Sample enrichment approaches:

  • Isolate mitochondrial fractions (differential centrifugation at 12,000g)

  • Process samples immediately with protease inhibitors to prevent degradation

  • Use mitochondrial markers (TOM20, VDAC) to normalize expression data

• Signal amplification techniques:

  • HRP-conjugated secondary antibodies with enhanced chemiluminescence

  • Tyramide signal amplification for immunohistochemistry

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

• Optimize antigen retrieval for fixed samples:

  • Heat-induced epitope retrieval (HIER) with citrate buffer (pH 6.0)

  • Enzymatic retrieval for heavily fixed tissues

  • Permeabilization optimization with Triton X-100 for ICC/IF applications

• Control strategies:

  • Include positive controls with known MTERF1 expression (colon cancer tissues show elevated expression )

  • Use MTERF1-overexpressing cell lines as reference standards

  • Perform parallel qPCR of mitochondrial transcripts (12S rRNA, 16S rRNA, ND1) to correlate with protein levels

How can researchers investigate the relationship between MTERF1 phosphorylation and its functional activity?

Evidence suggests that only the phosphorylated form of MTERF1 has transcription termination activity . To investigate this regulatory mechanism:

• Differential detection approach:

  • Compare results using phospho-specific versus total MTERF1 antibodies

  • Perform lambda phosphatase treatment before immunoblotting

  • Use Phos-tag SDS-PAGE to separate phosphorylated from non-phosphorylated forms

• Functional correlation studies:

  • Immunoprecipitate MTERF1 from different cellular states

  • Assess transcription termination activity using in vitro transcription assays

  • Compare results using LSP and HSP templates (MTERF1 terminates transcription from LSP more effectively)

• Site-specific analysis:

  • Use mass spectrometry to identify phosphorylation sites after immunoprecipitation

  • Generate phospho-mimetic and phospho-deficient MTERF1 mutants

  • Compare their binding activity and transcription termination function

• Experimental validation:

  • Treat cells with kinase inhibitors or phosphatase inhibitors

  • Monitor effects on MTERF1-dependent transcription termination

  • Correlate changes with mitochondrial function (membrane potential, ATP production)

How do researchers use MTERF1 antibodies to investigate its role in cancer progression?

MTERF1 shows significantly increased expression in colon cancer tissues compared to normal colorectal tissue . To investigate its role in cancer:

• Expression analysis protocols:

  • Compare MTERF1 levels between tumor and matched normal tissues using western blotting

  • Perform immunohistochemistry on tissue microarrays across cancer stages

  • Co-stain with proliferation markers (Ki-67) and apoptotic markers (cleaved caspase-3, PARP)

• Functional mechanisms investigation:

  • Examine effects on cell cycle (MTERF1 overexpression reduces G1 phase and increases S phase)

  • Analyze mtDNA copy number after MTERF1 modulation (qPCR of D-loop region normalized to 18S rDNA)

  • Measure mitochondrial transcript levels (12S rRNA, 16S rRNA, ND1, Cytb, ND6)

• Metabolic effects assessment:

  • Measure intracellular ATP levels (MTERF1 overexpression increases ATP by 2.4-fold)

  • Determine mitochondrial membrane potential using flow cytometry

  • Correlate MTERF1 expression with OXPHOS activity

• Experimental models approach:

  • Use gain-of-function (overexpression) and loss-of-function (RNAi knockdown) approaches

  • Validate antibody specificity in each model system

  • Track effects on cancer cell migration and invasion

These methods reveal MTERF1's potential oncogenic role through promoting cell cycle progression, inhibiting apoptosis, and enhancing mitochondrial function in cancer cells.

What experimental design considerations are important when investigating MTERF1's role in mitochondrial transcription regulation?

To properly investigate MTERF1's role in transcription regulation:

• Design transcription templates containing:

  • Light strand promoter (LSP) or heavy strand promoter (HSP)

  • MTERF1 binding site in correct orientation

  • Control regions (CSBII) to monitor specificity

• Assemble transcription reactions with:

  • Purified components (POLRMT, TFAM, TFB2M)

  • Varying concentrations of MTERF1 (titration approach)

  • Optional addition of TEFM to assess elongation effects

• Analyze transcription products:

  • Monitor full-length run-off transcripts

  • Identify MTERF1-dependent termination products

  • Compare termination efficiency between LSP and HSP templates

• Validation in cellular systems:

  • Use MTERF1 antibodies to confirm protein expression/knockdown

  • Measure levels of mitochondrial transcripts by qRT-PCR

  • Analyze both rRNAs (12S, 16S) and mRNAs (ND1, ND6, Cytb) to assess strand-specific effects

This systematic approach reveals MTERF1's directional specificity - it efficiently terminates L-strand transcription while showing minimal effect on H-strand transcription, especially in the presence of TEFM .

How can researchers differentiate between MTERF1's transcription termination and replication pause functions?

MTERF1 exhibits dual functionality in transcription termination and replication regulation . To distinguish these roles:

• Implement orientation-specific analysis:

  • Design constructs with MTERF1 binding sites in both orientations

  • Assess transcription termination efficiency from both directions

  • Analyze replication pausing in corresponding orientations

• Establish in vitro systems to separate functions:

  • Transcription assays with POLRMT, TFAM, TFB2M (± TEFM)

  • Replication assays with POLγ, mtSSB, TWINKLE

  • Add recombinant MTERF1 to both systems independently

• Analyze distinct products:

  • Terminated transcripts (RNA products)

  • Paused replication intermediates (DNA products)

  • Use denaturing gel electrophoresis for separation

• Perform time-course experiments:

  • Monitor appearance of terminated transcripts over time

  • Track accumulation of replication intermediates

  • Compare kinetics between processes

These approaches reveal that MTERF1's primary functions are to prevent antisense transcription over rRNA genes and to act as a directional contrahelicase in mtDNA replication .

What controls are essential when validating new MTERF1 antibodies for research applications?

When validating MTERF1 antibodies for research:

• Specificity controls:

  • Western blot confirmation of single band at expected molecular weight (39 kDa)

  • Peptide competition assays using the immunizing peptide (recombinant fragment within human MTERF1 aa 1-200)

  • Testing on MTERF1 knockout/knockdown samples

• Cross-reactivity assessment:

  • Testing against other MTERF family members (particularly MTERFD1, MTERFD3)

  • Validation across species (human, mouse, rat) if claiming cross-reactivity

  • Verification in tissues with known expression patterns

• Application-specific controls:

  • For ICC/IF: PFA-fixed, Triton X-100 permeabilized cells with nuclear counterstain

  • For WB: Include positive control cell lines (RT4, U-2 OS)

  • For IHC: Include both positive and negative tissue controls

• Quantitative validation:

  • Antibody titration to determine optimal concentration for each application

  • Signal-to-noise ratio assessment across concentration range

  • Reproducibility testing across different sample preparations

How should researchers interpret contradictory results when comparing data from different MTERF1 antibodies?

When facing contradictory results between different MTERF1 antibodies:

• Analyze antibody characteristics:

  • Compare epitope locations (N-terminal vs. C-terminal targeting)

  • Review clonality (monoclonal vs. polyclonal antibodies)

  • Check immunogen details (synthetic peptide vs. recombinant protein)

• Consider post-translational modifications:

  • Phosphorylation status (only phosphorylated MTERF1 may have transcription termination activity)

  • Other modifications that might mask epitopes

  • Protein conformation differences in various experimental conditions

• Evaluate technical variables:

  • Fixation/extraction methods affecting epitope accessibility

  • Buffer conditions influencing antibody binding

  • Detection systems with varying sensitivities

• Resolution approach:

  • Use multiple antibodies targeting different regions

  • Implement knockout/knockdown validation for each antibody

  • Consider epitope tagging of MTERF1 (FLAG, HA) for detection with tag antibodies

• Biological interpretation:

  • Different antibodies might reveal distinct MTERF1 populations or conformational states

  • Consider that contradictory results might reflect actual biological complexity

  • Correlate antibody detection with functional readouts (transcription, replication)

What methodological approaches help differentiate between mitochondrial and potential non-mitochondrial MTERF1 functions?

Although MTERF1 is primarily described as a mitochondrial protein, to investigate potential non-mitochondrial functions:

• Subcellular fractionation approach:

  • Separate mitochondrial, cytosolic, and nuclear fractions

  • Analyze MTERF1 distribution using validated antibodies

  • Include fraction-specific markers (TOM20 for mitochondria, GAPDH for cytosol, Lamin B for nucleus)

• Immunofluorescence co-localization:

  • Perform dual staining with MTERF1 antibodies and organelle markers

  • Use confocal microscopy with Z-stack imaging

  • Quantify co-localization coefficients (Pearson's, Mander's)

• Functional validation:

  • Compare effects of MTERF1 modulation on:

    • Mitochondrial transcription/replication

    • Nuclear gene expression

    • Cytosolic signaling pathways

• Inducible targeting approach:

  • Generate constructs with mitochondrial targeting sequence mutations

  • Create fusion proteins with organelle-specific targeting signals

  • Validate localization and function using antibodies against endogenous and tagged proteins

How can researchers use MTERF1 antibodies to investigate links between mitochondrial transcription and cancer metabolism?

MTERF1's increased expression in cancer suggests important metabolic implications . To investigate this connection:

• Multi-parameter profiling:

  • Perform co-staining of MTERF1 with metabolic markers (GLUT1, HK2, PKM2)

  • Analyze tissue microarrays across cancer types and stages

  • Correlate MTERF1 expression with metabolic phenotypes

• Functional metabolic assessment:

  • Measure MTERF1 expression/localization during metabolic stress

  • Combine MTERF1 immunodetection with Seahorse analysis of mitochondrial respiration

  • Track changes in MTERF1 binding (ChIP-qPCR) after metabolic interventions

• Cancer-specific research design:

  • Compare MTERF1 expression between glycolytic and oxidative tumors

  • Analyze effects of MTERF1 modulation on:

    • ATP production (MTERF1 overexpression increases ATP by 2.4-fold)

    • Mitochondrial membrane potential

    • Oxygen consumption rate

• Therapeutic targeting evaluation:

  • Monitor MTERF1 expression/function during treatment with metabolic inhibitors

  • Assess whether MTERF1 levels predict response to therapies targeting mitochondria

  • Investigate combination approaches targeting both MTERF1 and metabolic pathways

These approaches can help establish whether MTERF1's role in mitochondrial transcription termination contributes to the metabolic reprogramming in cancer cells.

What experimental design factors are critical when using MTERF1 antibodies to study the p-AMPK/mTOR pathway in relation to mitochondrial function?

To investigate MTERF1's relationship with the p-AMPK/mTOR pathway:

• Coordinate expression analysis:

  • Perform sequential or parallel western blotting for MTERF1, p-AMPK, mTOR

  • Use phospho-specific antibodies to track activation states

  • Include total protein controls for normalization

• Pathway manipulation approaches:

  • Treat cells with AMPK activators (AICAR, metformin) or inhibitors

  • Use mTOR modulators (rapamycin, Torin1)

  • Monitor MTERF1 expression, localization, and binding activity

• Genetic intervention strategies:

  • Combine MTERF1 knockdown/overexpression with p-AMPK/mTOR pathway analysis

  • Generate double knockdown/knockout models

  • Rescue experiments to establish causality

• Functional readouts:

  • Measure mitochondrial transcription after pathway modulation

  • Assess mtDNA copy number regulation

  • Monitor mitochondrial function (membrane potential, ATP production)

• Disease context relevance:

  • Compare findings between normal cells and cancer models

  • Analyze tissue samples for correlation between MTERF1 and pathway components

  • Consider metabolic status differences between tissues

This experimental approach helps establish whether MTERF1 functions within the p-AMPK/mTOR regulatory network controlling mitochondrial biogenesis and function.