ALDH1L2 Antibody

What is ALDH1L2 and what are its primary functions?

ALDH1L2 is a mitochondrial 10-formyltetrahydrofolate dehydrogenase that belongs to the aldehyde dehydrogenase family and ALDH1L subfamily. It catalyzes the NADP(+)-dependent conversion of 10-formyltetrahydrofolate to tetrahydrofolate and carbon dioxide. This enzymatic reaction is an important source of NADPH in mitochondria, linking ALDH1L2 to cellular redox balance and cancer metastasis. ALDH1L2 ensures adequate supply of substrates required for DNA synthesis and repair, suggesting its importance in maintaining genomic stability and nucleotide balance . Unlike its cytosolic homolog ALDH1L1, ALDH1L2 is frequently overexpressed in cancer cells, indicating its potential role in tumorigenesis and cancer progression .

What applications are ALDH1L2 antibodies suitable for?

ALDH1L2 antibodies are suitable for multiple research applications, including:

• Western Blot (WB): Typically used at dilutions of 1:500-1:3000

• Immunohistochemistry (IHC): Recommended dilutions of 1:20-1:200

• Immunofluorescence (IF)/Immunocytochemistry (ICC): Effective at dilutions of 1:200-1:800

• ELISA: For quantitative protein detection

These antibodies have been validated for reactivity with human, mouse, and rat samples, with cited reactivity also in pig samples . It's important to note that optimal dilutions may be sample-dependent and should be determined experimentally for each testing system.

What is the molecular weight of ALDH1L2 and how does it appear on Western blots?

ALDH1L2 has a calculated molecular weight of 102 kDa (923 amino acids). On Western blots, ALDH1L2 is typically observed at either 102 kDa or 89 kDa, depending on the isoform detected . The ALDH1L2 gene has three transcriptional variants, which can result in different molecular weights when visualized by Western blotting. When performing Western blot analysis, researchers should be aware of these potential size variations to correctly identify ALDH1L2 bands .

How should I optimize antibody conditions for ALDH1L2 detection in IHC applications?

For optimal ALDH1L2 detection in immunohistochemistry applications:

• Antigen retrieval: Use TE buffer at pH 9.0 (primary recommendation) or alternatively, citrate buffer at pH 6.0

• Initial dilution testing: Begin with 1:50 for strong signals and adjust to 1:100-1:200 for reduced background

• Positive control tissues: Human pancreas and heart tissues have been validated as positive controls

• Incubation conditions: Typically overnight incubation at 4°C provides optimal results

• Detection system: Use a detection system compatible with rabbit IgG primary antibodies

• Counterstaining: Hematoxylin counterstaining for 1-2 minutes provides good nuclear contrast without obscuring specific staining

Optimization should include a titration series to determine the optimal antibody concentration for your specific tissue samples, as signal intensity can vary between different tissue types and fixation methods.

What controls should I include when using ALDH1L2 antibodies for research applications?

A robust experimental design with ALDH1L2 antibodies should include the following controls:

• Positive tissue controls: Human pancreas and heart tissues for IHC; mouse and rat pancreas tissues for Western blot

• Negative controls: Primary antibody omission and isotype controls

• ALDH1L2 knockdown/knockout samples: Including CRISPR-generated ALDH1L2 knockout samples as negative controls

• Blocking peptide controls: Pre-incubating the antibody with blocking peptide to confirm specificity

• Comparison with alternative antibody clones: Using a different ALDH1L2 antibody to verify staining patterns

For knockout validation, CRISPR-Cas9 targeting either exon 1 or exon 3 of ALDH1L2 has been successfully used to generate ALDH1L2 knockout cell lines, as demonstrated in U251 glioblastoma studies .

How can I effectively use ALDH1L2 antibodies to study protein-protein interactions?

To study ALDH1L2 protein interactions:

• Immunoprecipitation approach: Use anti-ALDH1L2 antibodies conjugated to protein A/G beads to pull down ALDH1L2 and its interacting partners

• Co-immunoprecipitation verification: Perform reverse co-IP using antibodies against suspected interacting proteins (e.g., SIRT3 )

• Protein crosslinking: Consider using crosslinking reagents to stabilize transient interactions

• Mass spectrometry analysis: Analyze immunoprecipitated complexes by mass spectrometry to identify novel interacting partners, as demonstrated in studies identifying SIRT3 as an ALDH1L2-associated protein

• Proximity ligation assay: Visualize protein interactions in situ using two different primary antibodies and proximity probes

When studying ALDH1L2 interactions, consider treating cells with nicotinamide (NAM), a pan-sirtuin family inhibitor, which enhances ALDH1L2 acetylation and may affect interaction profiles .

How does ALDH1L2 expression correlate with cancer progression and how can antibodies help study this relationship?

ALDH1L2 expression shows significant correlation with cancer progression and can be studied using antibodies through:

• Tissue microarray analysis: Antibody staining of cancer and matched normal tissue microarrays to quantify expression differences

• Correlation with patient outcomes: Analyzing ALDH1L2 expression in relation to patient survival data

What is the significance of ALDH1L2 acetylation and how can researchers investigate this post-translational modification?

ALDH1L2 acetylation represents a critical post-translational modification that regulates its enzymatic activity. To investigate ALDH1L2 acetylation:

• Acetylation detection: Immunoprecipitate ALDH1L2 using specific antibodies and probe with anti-acetyl-lysine antibodies

• Site-specific mutants: Generate K70R (deacetylation mimic) and K70Q (acetylation mimic) ALDH1L2 mutants to study functional effects

• Deacetylase modulation: Treat cells with nicotinamide (NAM) to inhibit sirtuin deacetylases and enhance ALDH1L2 acetylation

• Activity assays: Compare enzymatic activity between wild-type and mutant (K70R/K70Q) ALDH1L2

Research has shown that ALDH1L2 is acetylated at lysine 70 (K70), and this acetylation decreases its enzymatic activity. SIRT3, an NAD+-dependent deacetylase, interacts with ALDH1L2 and regulates its acetylation status . K70 is highly conserved across several mammalian species, highlighting the evolutionary importance of this regulatory mechanism .

How can researchers utilize ALDH1L2 antibodies to study its role in cellular redox balance and cancer metabolism?

To investigate ALDH1L2's role in redox balance and cancer metabolism:

• Immunofluorescence co-localization: Use ALDH1L2 antibodies with mitochondrial markers to study subcellular localization

• Expression manipulation: Combine antibody detection with ALDH1L2 knockdown/overexpression to monitor metabolic changes

• Metabolic stress response: Analyze ALDH1L2 expression/localization under oxidative stress conditions

• Metabolite analysis: Correlate ALDH1L2 expression levels with measurements of NADPH/NADP+, GSH/GSSG ratios, and ROS levels

Studies have shown that ALDH1L2 plays a critical role in maintaining NADPH production in mitochondria, which affects cellular redox balance. ALDH1L2 wild-type cells display higher NADPH/NADP+ and GSH/GSSG ratios compared to ALDH1L2 K70Q (acetylation mimic) cells, along with lower ROS levels and reduced apoptosis . Additionally, ALDH1L2 knockout in glioblastoma cells increases oxidative stress and suppresses methionine dependency, reducing tumor sphere formation .

What are common issues encountered when using ALDH1L2 antibodies and how can they be resolved?

Common issues with ALDH1L2 antibodies and their solutions:

When troubleshooting, remember that ALDH1L2 is primarily localized in mitochondria, so proper sample preparation to preserve mitochondrial integrity is essential for accurate detection.

How should researchers interpret variations in ALDH1L2 expression patterns across different tissue types?

When interpreting ALDH1L2 expression across tissues:

• Establish baseline expression: Compare your findings with established expression patterns (e.g., pancreas and heart tissues show reliable ALDH1L2 expression)

• Consider metabolic status: ALDH1L2 expression can correlate with tissue metabolic activity

• Evaluate cellular heterogeneity: Within complex tissues, expression may vary between cell types

• Differentiate normal vs. pathological expression: In cancer studies, compare with matched non-cancerous tissues

• Account for technical variables: Tissue processing, fixation, and antibody lot variations can affect staining intensity

Research has shown that ALDH1L2 expression is higher in CRC cell lines compared to normal colon cell lines, with HCT116, CaCO2, and SW480 cell lines showing relatively high expression . When analyzing patient samples, consider that more than half of CRC samples (62.2%) display elevated ALDH1L2 expression compared to matched normal tissues .

How can researchers validate ALDH1L2 antibody specificity for their experimental system?

To validate ALDH1L2 antibody specificity:

• Genetic validation: Use CRISPR/Cas9 ALDH1L2 knockout cells as negative controls; confirmed knockout targeting exon 1 or exon 3 has been established in research models

• siRNA knockdown validation: Confirm reduced signal after ALDH1L2 siRNA treatment; knockdown in HCT116, CaCO2, and SW480 cell lines has been successfully performed

• Peptide competition: Pre-incubate antibody with immunizing peptide to block specific binding

• Multiple antibody comparison: Compare staining patterns using antibodies targeting different ALDH1L2 epitopes

• Western blot correlation: Confirm that IHC or IF findings correlate with Western blot results from the same samples

Validation should include positive controls such as human fetal kidney, heart, and liver lysates, which have been shown to express detectable levels of ALDH1L2 .

How is ALDH1L2 research evolving and what new applications of ALDH1L2 antibodies are emerging?

Recent advances in ALDH1L2 research include:

• Cancer metabolism studies: Emerging evidence links ALDH1L2 to cancer-specific metabolic adaptations, particularly in colorectal cancer and glioblastoma

• Post-translational modifications: Research has revealed acetylation as a key regulatory mechanism for ALDH1L2 activity, with K70 identified as a critical acetylation site

• Chemosensitivity modulation: ALDH1L2 acetylation status affects sensitivity to chemotherapeutic agents like 5-Fu, suggesting potential for combination therapies

• Redox balance regulation: ALDH1L2's role in maintaining NADPH/NADP+ ratios positions it as a key regulator of cellular oxidative stress

Future applications of ALDH1L2 antibodies may include prognostic biomarker development, patient stratification for targeted therapies, and monitoring treatment responses in cancer patients.

What methodological advances are improving the detection and quantification of ALDH1L2 in complex biological samples?

Methodological improvements for ALDH1L2 detection include:

• Multiplex immunofluorescence: Simultaneous detection of ALDH1L2 with other markers to study pathway interactions

• Automated image analysis: Algorithm-based quantification of staining intensity and subcellular localization

• Single-cell analysis: Combination with single-cell technologies to assess heterogeneity within populations

• Proximity ligation assays: Enhanced detection of protein-protein interactions involving ALDH1L2

• Mass cytometry: Highly multiplexed analysis of ALDH1L2 alongside dozens of other cellular markers

These advances enable more precise quantification of ALDH1L2 expression and localization in heterogeneous samples such as tumor tissues, potentially improving diagnostic and prognostic applications.