MTHFD1L Antibody

What is MTHFD1L and why is it important in cellular metabolism?

MTHFD1L is a mitochondrial enzyme that catalyzes the final step in the mitochondrial compartment of the folate cycle, generating formate that subsequently enters the cytoplasmic compartment. This enzyme plays a critical role in linking mitochondrial and cytoplasmic one-carbon metabolism . MTHFD1L contributes significantly to NADPH production, which serves as a major cellular antioxidant and helps combat oxidative stress .

The importance of MTHFD1L extends beyond basic metabolism: knockout studies have demonstrated that MTHFD1L-knockout mice exhibit fetal growth delay, neural tube and craniofacial abnormalities, and embryonic lethality . In humans, MTHFD1L polymorphisms are associated with neural tube defects, coronary artery disease, and Alzheimer's disease .

Most significantly, MTHFD1L has emerged as a key player in cancer metabolism. It is overexpressed in multiple cancer types including hepatocellular carcinoma (HCC), esophageal cancer, stomach adenocarcinoma, bladder cancer, lung adenocarcinoma, breast cancer, colon adenocarcinoma, kidney renal cell carcinoma, and prostate adenocarcinoma . This overexpression correlates with aggressive clinicopathological features and poorer patient survival, particularly in HCC .

What applications are MTHFD1L antibodies validated for?

MTHFD1L antibodies have been validated for multiple experimental applications:

Different antibodies target distinct epitopes within MTHFD1L. For example, Abcam ab116615 targets human MTHFD1L within amino acids 550-600, while ab221925 targets within amino acids 200-350 . This diversity allows researchers to select antibodies based on their specific experimental requirements and the protein domain of interest.

What species reactivity is available for MTHFD1L antibodies?

Researchers working with different model systems can select MTHFD1L antibodies based on their species reactivity:

When selecting an antibody for cross-species applications, it's important to verify that it has been validated for your species of interest . Some antibodies may work with additional species not explicitly listed due to sequence homology in the target epitope region, but this should be experimentally confirmed.

What is the molecular weight of MTHFD1L protein and what band should I expect in Western blot?

MTHFD1L has a calculated molecular weight of approximately 106 kDa based on its 978 amino acid sequence . In Western blot experiments, the observed molecular weight typically ranges from 106-110 kDa:

• Abcam ab116615 shows a band at approximately 110 kDa in human cerebellum tissue lysate

• Proteintech antibodies report an observed molecular weight of 106 kDa

• Sigma's antibody HPA074911 can detect MTHFD1L at the expected molecular weight with recommended dilutions of 0.04-0.4 μg/mL

When performing Western blot analysis, include appropriate positive controls such as HEK-293, HeLa, or HepG2 cell lysates, which are known to express MTHFD1L . Minor variations in observed molecular weight may occur due to post-translational modifications or different isoforms of the protein.

How can MTHFD1L antibodies be used to study cancer metabolism?

MTHFD1L has emerged as a significant player in cancer metabolism, particularly in relation to NADPH production and oxidative stress responses. Researchers can utilize MTHFD1L antibodies in several advanced cancer research applications:

• Expression profiling across cancer types:

  • IHC analysis of tissue microarrays containing multiple cancer types

  • Western blot comparison of MTHFD1L expression across cancer cell lines

  • Correlation with clinicopathological features as demonstrated in HCC patients

• Metabolic phenotype characterization:

  • Combine MTHFD1L expression analysis with measurements of NADPH/NADP+ ratios

  • Assess ROS levels in relation to MTHFD1L expression

  • Study the impact of folate cycle modulation on cancer cell metabolism

• Therapeutic targeting studies:

  • Monitor MTHFD1L expression changes after treatment with antifolate drugs like methotrexate

  • Investigate drug combinations (e.g., methotrexate and sorafenib showed synergistic effects in HCC)

  • Evaluate MTHFD1L as a biomarker for treatment response

Research has shown that MTHFD1L knockdown leads to:

• Reduced NADPH/NADP+ ratio

• Increased ROS accumulation

• Repressed cancer cell proliferation

• G1 phase cell cycle arrest

• Enhanced sensitivity to targeted therapies such as sorafenib

These findings highlight the potential of MTHFD1L as both a biomarker and therapeutic target in cancer, making antibodies against this protein valuable tools in cancer metabolism research.

What controls should be included when validating MTHFD1L knockdown or knockout?

Proper validation of MTHFD1L knockdown or knockout is critical for ensuring experimental rigor. Based on available research, the following controls and validation methods are recommended:

When conducting immunoblot validation, use at least two different antibodies targeting distinct epitopes of MTHFD1L to confirm specificity. Additionally, include appropriate loading controls and quantify the reduction in MTHFD1L expression relative to controls.

According to research findings, successful MTHFD1L knockdown should result in not only reduced protein expression but also functional consequences including decreased NADPH/NADP+ ratio, elevated ROS levels, and G1 phase cell cycle arrest . These functional readouts provide crucial validation beyond simple protein expression analysis.

How do different sample preparation methods affect MTHFD1L detection?

MTHFD1L is primarily localized in mitochondria, which makes sample preparation methodology particularly important for optimal detection:

• Tissue samples:

  • Fresh frozen tissues typically yield better results than formalin-fixed paraffin-embedded (FFPE) samples for Western blot

  • For FFPE samples, extended antigen retrieval may be necessary for IHC detection

  • Recommended antigen retrieval: TE buffer pH 9.0 or citrate buffer pH 6.0

• Cell lysis for Western blot:

  • RIPA buffer has been successfully used for MTHFD1L extraction

  • Include protease inhibitors to prevent degradation

  • Consider mitochondrial enrichment protocols for enhanced detection

  • Protein loading: 35-50 μg of total protein per lane is recommended

• Fixation for immunofluorescence:

  • PFA fixation (4%) followed by Triton X-100 permeabilization works well

  • Co-staining with mitochondrial markers helps confirm proper localization

  • Methanol fixation can be an alternative for some applications

• Subcellular fractionation:

  • Separate mitochondrial and cytosolic fractions to study compartment-specific expression

  • Include compartment-specific markers (e.g., VDAC for mitochondria, GAPDH for cytosol)

  • Compare MTHFD1L levels in whole cell lysates versus mitochondrial fractions

When investigating MTHFD1L in cancer specimens, it's important to note that overexpression has been documented in 50.59% (43/85) of HCC patients by at least 2-fold . This suggests that sample preparation methods should be optimized to detect varying expression levels across different specimens.

What are the best protocols for studying MTHFD1L's role in the folate cycle?

MTHFD1L plays a crucial role in linking mitochondrial and cytoplasmic components of the folate cycle. To comprehensively study its role, consider the following integrated approaches:

• Expression analysis with metabolic profiling:

  • Combine MTHFD1L antibody detection with folate metabolite measurement

  • Correlate MTHFD1L levels with NADPH/NADP+ ratios and ROS measurements

  • Study co-expression with other folate cycle enzymes (MTHFD1, MTHFD2, SHMT1/2)

• Functional perturbation experiments:

  • Use siRNA/shRNA to knockdown MTHFD1L followed by metabolic profiling

  • Combine with antifolate drugs (e.g., methotrexate) to assess synergistic effects

  • Measure formate production, which MTHFD1L generates as the final step in the mitochondrial folate pathway

• Subcellular localization studies:

  • Co-immunofluorescence of MTHFD1L with mitochondrial markers

  • Fractionation studies to examine compartment-specific expression

  • Investigate potential shuttling between compartments under metabolic stress

• Metabolic flux analysis:

  • Combine MTHFD1L expression analysis with stable isotope tracing

  • Assess impact of MTHFD1L modulation on one-carbon metabolic flux

  • Study how MTHFD1L levels affect purine synthesis and methylation reactions

Research has shown that the folate cycle is a major source of NADPH, and MTHFD1L plays a critical role in maintaining this cycle . A recent meta-study including microarray data from more than 1,900 tumor tissues across 19 different cancer types identified the mitochondrial 1-carbon metabolism as the most deregulated metabolic pathway in human cancers , highlighting the importance of MTHFD1L in cancer metabolism.

What is the recommended protocol for MTHFD1L Western blot analysis?

For optimal MTHFD1L detection by Western blot, follow this detailed protocol based on validated antibody applications:

Sample Preparation:

• Lyse cells or tissues in RIPA buffer with protease inhibitors

• Determine protein concentration (BCA or Bradford assay)

• Prepare samples with 4X Laemmli buffer + DTT or β-mercaptoethanol

• Heat samples at 95°C for 5 minutes

Gel Electrophoresis:

• Load 35-50 μg protein per lane on 8-10% SDS-PAGE gel

• Include molecular weight markers (75-150 kDa range)

• Run gel at 80-100V until dye front reaches bottom

Transfer:

• Transfer to PVDF membrane (0.45 μm) at 100V for 60-90 minutes or 30V overnight at 4°C

• Verify transfer with Ponceau S staining

Immunoblotting:

• Block membrane with 5% non-fat milk or BSA in TBST for 1 hour at room temperature

• Incubate with primary antibody in blocking buffer:

  • Abcam ab116615: 0.1 μg/mL

  • Abcam ab221925: 1/100 dilution

  • Proteintech 16113-1-AP: 1:2000-1:10000

  • Proteintech 68321-1-Ig: 1:5000-1:50000

  • Sigma HPA074911: 0.04-0.4 μg/mL

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

• Wash 3×10 minutes with TBST

• Incubate with appropriate HRP-conjugated secondary antibody (1:5000-1:10000) for 1 hour

• Wash 3×10 minutes with TBST

Detection:

• Apply ECL substrate and develop using film or digital imager

• Expected band: 106-110 kDa

Positive Controls:

• Human: HEK-293, HeLa, HepG2, MCF7, RT4 cells; cerebellum tissue

• Mouse: Brain tissue, NIH3T3 cells

• Rat: Testis tissue

The detection strategy may need adjustment based on expression levels. For high-expressing samples like cancer cell lines, brief exposures may be sufficient, while normal tissues might require longer exposure times or more sensitive detection methods.

What is the optimized protocol for MTHFD1L immunohistochemistry?

For successful MTHFD1L immunohistochemistry in tissue sections, follow this optimized protocol based on validated antibody applications:

Tissue Preparation:

• Fix tissues in 10% neutral buffered formalin

• Process and embed in paraffin

• Section at 4-5 μm thickness and mount on adhesive slides

Deparaffinization and Rehydration:

• Xylene: 2 × 5 minutes

• 100% ethanol: 2 × 3 minutes

• 95%, 80%, 70% ethanol: 3 minutes each

• Wash in distilled water

Antigen Retrieval:

• Recommended buffer: TE buffer pH 9.0

• Alternative: Citrate buffer pH 6.0

• Heat in pressure cooker or microwave until boiling

• Maintain at sub-boiling temperature for 15-20 minutes

• Cool to room temperature (20 minutes)

Staining Procedure:

• Quench endogenous peroxidase: 3% H₂O₂ for 10 minutes

• Wash in PBS/TBS: 2 × 5 minutes

• Block with 5-10% normal serum for 30-60 minutes

• Apply primary antibody diluted in blocking solution:

  • Proteintech 16113-1-AP: 1:50-1:500

  • Proteintech 68321-1-Ig: 1:250-1:1000

  • Abcam ab116615: Follow manufacturer's recommendation

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

• Wash in PBS/TBS: 3 × 5 minutes

• Apply appropriate HRP-conjugated secondary antibody for 30-60 minutes

• Wash in PBS/TBS: 3 × 5 minutes

• Develop with DAB substrate kit (3-5 minutes or until optimal color develops)

• Counterstain with hematoxylin (30 seconds)

• Dehydrate, clear, and mount with permanent mounting medium

Controls:

• Positive tissue control: Liver or liver cancer tissue (known to express MTHFD1L)

• Negative control: Omit primary antibody

• If available, MTHFD1L knockdown tissue as specificity control

Scoring and Interpretation:
For cancer studies, evaluate MTHFD1L expression considering:

• Staining intensity (0-3+)

• Percentage of positive cells

• Subcellular localization (primarily mitochondrial)

• Comparison between tumor and adjacent normal tissue

Research has shown that MTHFD1L overexpression in HCC correlates with aggressive features including tumor microsatellite formation, venous invasion, and advanced tumor stages . This makes IHC analysis particularly valuable for clinicopathological correlation studies.

What are the optimal conditions for MTHFD1L immunofluorescence staining?

For effective visualization of MTHFD1L by immunofluorescence, follow this optimized protocol based on validated procedures:

Cell Preparation:

• Culture cells on glass coverslips to 60-80% confluence

• Rinse gently with PBS (pre-warmed to 37°C)

Fixation and Permeabilization:

• Fix with 4% paraformaldehyde in PBS for 15-20 minutes at room temperature

• Wash 3× with PBS (5 minutes each)

• Permeabilize with 0.1-0.5% Triton X-100 in PBS for 10 minutes

• Wash 3× with PBS (5 minutes each)

Immunostaining:

• Block with 1-5% BSA or normal serum in PBS for 30-60 minutes

• Incubate with primary antibody diluted in blocking solution:

  • Abcam ab221925: 4 μg/ml

  • Proteintech 16113-1-AP: 1:50-1:500

  • Sigma HPA074911: 0.25-2 μg/mL

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

• Wash 3× with PBS (5 minutes each)

• Incubate with fluorophore-conjugated secondary antibody (1:200-1:1000) for 1 hour at room temperature

• Wash 3× with PBS (5 minutes each)

• Counterstain nuclei with DAPI (1 μg/mL) for 5 minutes

• Mount with anti-fade mounting medium

Co-staining for Mitochondria:

• Include mitochondrial marker antibody (e.g., Tom20, COX IV) or

• Stain with MitoTracker (pre-incubation before fixation)

• Use different fluorophores for MTHFD1L and mitochondrial markers

Recommended Cell Types for Positive Control:

• HepG2 cells

• MCF7 cells (validated in search results)

• HeLa cells

Imaging and Analysis:

• Use confocal microscopy for optimal resolution of mitochondrial structures

• Collect Z-stack images to fully visualize mitochondrial network

• Expected pattern: Punctate/reticular staining consistent with mitochondrial localization

• Analyze colocalization with mitochondrial markers

• Compare expression levels between different cell types or treatment conditions

Since MTHFD1L is primarily localized to mitochondria where it functions in the folate cycle, proper visualization of its subcellular localization is crucial for understanding its functional role in normal and disease states.

How can I optimize immunoprecipitation using MTHFD1L antibodies?

For successful immunoprecipitation (IP) of MTHFD1L and its potential interaction partners, follow this optimized protocol:

Lysate Preparation:

• Harvest cells (HeLa cells recommended as positive control)

• Lyse cells in a gentle IP buffer:

  • 50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% NP-40 or 0.5% Triton X-100

  • Supplement with protease and phosphatase inhibitors

  • Incubate on ice for 30 minutes with occasional mixing

• Centrifuge at 12,000×g for 15 minutes at 4°C

• Collect supernatant and determine protein concentration

• Use 1-3 mg total protein per IP reaction

Pre-clearing (Optional but Recommended):

• Add 20 μL Protein A/G beads to lysate

• Rotate for 1 hour at 4°C

• Remove beads by centrifugation

Immunoprecipitation:

• Add MTHFD1L antibody to pre-cleared lysate:

  • Proteintech 16113-1-AP: 0.5-4.0 μg per 1-3 mg lysate

• Incubate overnight at 4°C with gentle rotation

• Add 30-50 μL Protein A/G beads

• Rotate for 2-4 hours at 4°C

• Collect beads by centrifugation (2000×g for 2-3 minutes)

• Wash beads 4× with cold IP buffer

• Optional additional wash with high salt buffer (300 mM NaCl)

Elution and Analysis:

• Add 40-50 μL 2× Laemmli buffer

• Heat at 95°C for 5 minutes

• Centrifuge and collect supernatant

• Analyze by SDS-PAGE and Western blot

Controls:

• Input: 5-10% of lysate used for IP

• IgG control: Same amount of non-specific IgG matching the host species of the MTHFD1L antibody

• Negative control: Lysate from MTHFD1L knockdown cells

• Reverse IP: Use antibodies against suspected interaction partners to IP, then blot for MTHFD1L

Expected Interaction Partners:
Based on MTHFD1L's role in folate metabolism, potential interaction partners may include:

• Other folate cycle enzymes (MTHFD1, MTHFD2, SHMT1/2)

• Mitochondrial proteins involved in one-carbon metabolism

• Proteins participating in NADPH generation or ROS regulation

Successful IP of MTHFD1L not only confirms the specificity of the antibody but also provides a powerful tool for identifying its protein interaction network, which may reveal new insights into its function in normal and disease states.

How do I troubleshoot weak or non-specific MTHFD1L antibody signals?

When facing difficulties with MTHFD1L detection, consider these problem-solving approaches:

Specific Optimization Strategies:

• For Western blot:

  • Try reducing gel percentage (8%) for better resolution of the ~106 kDa protein

  • Use freshly prepared transfer buffer with 20% methanol

  • Increase transfer time for large proteins

  • Consider enhanced detection systems for low expression

• For IHC:

  • Test both citrate (pH 6.0) and TE (pH 9.0) buffers for antigen retrieval

  • Optimize antigen retrieval time (15-30 minutes)

  • Include mild detergent (0.1% Tween-20) in antibody diluent

  • Use polymer detection systems for increased sensitivity

• For IF:

  • Compare different fixation methods (PFA vs. methanol)

  • Optimize permeabilization conditions (concentration and time)

  • Reduce autofluorescence with sodium borohydride treatment

  • Use confocal microscopy for better resolution of mitochondrial staining

Always include appropriate positive controls such as HCC tissues or HepG2 cells, which are known to express high levels of MTHFD1L .

What is the significance of MTHFD1L in clinical cancer research?

MTHFD1L has emerged as a significant player in cancer biology with potential clinical implications:

These findings highlight MTHFD1L as a promising target for cancer diagnosis, prognosis, and therapy. MTHFD1L antibodies are essential tools for investigating these clinical applications through tissue-based studies, mechanism exploration, and biomarker development.

How does MTHFD1L expression vary across normal and disease tissues?

Understanding the tissue distribution pattern of MTHFD1L is crucial for interpreting experimental results:

• Normal tissue expression:

  • MTHFD1L shows variable expression across normal tissues

  • Higher expression is observed in:

    • Liver tissue (important for one-carbon metabolism)

    • Embryonic tissues (critical for development)

    • Testis tissue (used as positive control in some antibody validations)

  • Lower expression in most other adult tissues

• Developmental significance:

  • MTHFD1L knockout mice exhibit:

    • Fetal growth delay

    • Neural tube defects

    • Craniofacial abnormalities

    • Embryonic lethality

  • This indicates essential roles in embryogenesis and development

• Cancer-specific alterations:

  • Significant overexpression in multiple cancer types as mentioned previously

  • 50.59% of HCC patients show at least 2-fold overexpression

  • Expression correlates with tumor aggressiveness and patient outcomes

• Other disease associations:

  • MTHFD1L polymorphisms are associated with:

    • Neural tube defects

    • Coronary artery disease

    • Alzheimer's disease

• Subcellular localization:

  • Primarily mitochondrial localization

  • Functions at the interface between mitochondrial and cytoplasmic compartments of folate metabolism

  • Critical for maintaining compartmentalized one-carbon metabolism

When designing experiments to study MTHFD1L, consider these tissue-specific expression patterns for selecting appropriate controls and interpreting results. The differential expression between normal and cancer tissues makes MTHFD1L particularly interesting as a potential biomarker and therapeutic target.

What are the key considerations for successful MTHFD1L antibody-based research?

Successful research using MTHFD1L antibodies requires careful consideration of several factors:

• Antibody selection:

  • Choose antibodies validated for your specific application (WB, IHC, IF, IP)

  • Verify species reactivity matches your experimental model

  • Consider using antibodies targeting different epitopes for confirmation

  • Review validation data and publications using the antibody

• Experimental design:

  • Include appropriate positive controls (HepG2, HeLa cells; liver tissue)

  • Use negative controls (knockout/knockdown samples, antibody omission)

  • Consider MTHFD1L's mitochondrial localization in sample preparation

  • Optimize protocols based on expected expression levels

• Data interpretation:

  • Expected molecular weight: 106-110 kDa

  • Anticipate higher expression in cancer vs. normal tissues

  • Consider variations in expression across different normal tissues

  • Correlate with functional readouts (NADPH/NADP+ ratio, ROS levels)

• Technical optimizations:

  • Antigen retrieval (TE buffer pH 9.0 recommended)

  • Antibody dilution ranges specific to each application

  • Subcellular fractionation for enhanced detection

  • Co-staining with mitochondrial markers for localization studies

By integrating these considerations, researchers can effectively utilize MTHFD1L antibodies to advance understanding of this important metabolic enzyme in normal development, cancer biology, and other disease contexts.

What future research directions involving MTHFD1L antibodies are most promising?

Several promising research directions can be pursued using MTHFD1L antibodies:

• Cancer metabolism investigations:

  • Comprehensive profiling of MTHFD1L expression across cancer types and stages

  • Correlation with metabolic signatures and oxidative stress markers

  • Investigation of MTHFD1L as a predictive biomarker for antifolate therapy response

  • Development of MTHFD1L-targeted therapeutics

• Mechanistic studies:

  • Identification of MTHFD1L interaction partners through IP-MS approaches

  • Investigation of post-translational modifications regulating MTHFD1L activity

  • Analysis of MTHFD1L's role in compartmentalized folate metabolism

  • Study of MTHFD1L regulation under various stress conditions

• Clinical applications:

  • Development of MTHFD1L-based prognostic scoring systems for cancers

  • Evaluation of MTHFD1L as a circulating biomarker in liquid biopsies

  • Assessment of MTHFD1L's predictive value for treatment response

  • Correlation with genetic polymorphisms in cancer susceptibility

• Emerging therapeutic approaches:

  • Screening for specific MTHFD1L inhibitors

  • Testing combination therapies targeting MTHFD1L and oxidative stress pathways

  • Exploring synthetic lethality with other metabolic pathways

  • Developing MTHFD1L-based immunotherapy approaches