ALDH1A2 Antibody

What is ALDH1A2 and what is its biological significance?

ALDH1A2 (aldehyde dehydrogenase family 1 member A2), also known as RALDH2 (retinal dehydrogenase 2), belongs to the aldehyde dehydrogenase family. It functions as a cytosolic homotetramer (56.7 kDa subunits) and exhibits complex expression patterns throughout embryonic development . ALDH1A2 plays a critical role in retinoic acid synthesis, which regulates numerous developmental and physiological processes. Research has shown that ALDH1A2 knockout in mice is embryonic lethal, highlighting its essential role in development . Recent studies have also revealed that ALDH1A2 may have anti-inflammatory functions through its role in retinoic acid production .

What are the validated applications for ALDH1A2 antibodies?

ALDH1A2 antibodies have been validated for multiple research applications, with specific validation depending on the antibody clone. Common validated applications include:

When designing experiments, researchers should consult the specific validation data for their antibody of choice, as reactivity can vary between clones and applications .

What are the recommended dilutions for different experimental applications?

Optimal dilutions vary by application and specific antibody. The following table summarizes recommended dilutions based on current research:

It is strongly recommended that researchers titrate the antibody in their specific testing system to obtain optimal results, as sample type and experimental conditions can significantly affect performance .

What species reactivity do ALDH1A2 antibodies demonstrate?

ALDH1A2 antibodies show varying species reactivity depending on the clone. Based on the available data:

• Polyclonal antibody (13951-1-AP): Shows reactivity with human, mouse, and rat samples

• Monoclonal antibody (E6O6Q, #55481): Primarily shows reactivity with human samples

Researchers should verify cross-reactivity when working with species not explicitly validated by the manufacturer to ensure experimental validity .

How can ALDH1A2 antibodies be used to study stem cell populations?

ALDH activity serves as an important marker for stem cells, both normal and malignant . When using ALDH1A2 antibodies to study stem cell populations:

• Flow cytometry approach: ALDH1A2 antibodies can be used in combination with the Aldefluor assay, which measures ALDH activity in viable cells. The Aldefluor assay has become a standard method for studying ALDH expression in hematopoietic cells .

• Methodological considerations: When studying ALDH1A2 in stem cells, researchers should:

  • Include proper negative controls using ALDH inhibitors like DEAB (diethylaminobenzaldehyde)

  • Use multiparameter flow cytometry to correlate ALDH1A2 expression with other stem cell markers

  • Consider cell sorting of ALDH-high and ALDH-low populations for functional studies

• Data interpretation: Studies have confirmed that ALDH activity correlates well with hematopoietic stem cell activity. Cell lines with higher ALDH1A2 expression exhibit increased proliferation rates and clonal efficiency, consistent with stem cell characteristics .

What is ALDH1A2's role in drug resistance mechanisms?

Research demonstrates that ALDH1A2 overexpression contributes to drug resistance in multiple cell lines. Key findings include:

• Enhanced chemoresistance: Cell lines overexpressing ALDH1A2 show increased resistance to chemotherapeutic agents including 4-hydroperoxycyclophosphamide and doxorubicin .

• Experimental approach: To study ALDH1A2's role in drug resistance:

  • Establish cell lines with controlled ALDH1A2 expression levels (overexpression or knockdown)

  • Use MTT or similar viability assays to measure drug toxicity responses

  • Compare IC50 values between wild-type and ALDH1A2-modified cells

  • Include both ALDH substrate drugs (like cyclophosphamide) and non-substrate drugs (like doxorubicin) in studies

• Mechanistic insights: While 4-HC is a known substrate for certain ALDH isoforms, the protection against doxorubicin suggests ALDH1A2 may confer resistance through indirect mechanisms beyond direct drug metabolism .

How do genetic variants of ALDH1A2 impact protein function and disease?

Genetic variants of ALDH1A2 have been associated with disease risk and progression:

• Expression differences: RNA-sequencing of cartilage from osteoarthritis patients revealed gene expression differences between individuals with low-risk and high-risk ALDH1A2 variants .

• Experimental approaches: To study ALDH1A2 variants:

  • Perform genotyping to identify variant carriers

  • Use RNA-sequencing to assess differential gene expression

  • Include patient gender and other relevant covariates in the analysis model

  • Validate findings with additional methodologies such as qPCR or protein expression analysis

• Functional consequences: Studies suggest ALDH1A2 variants may influence the anti-inflammatory role of retinoic acid in diseases like osteoarthritis, affecting associated gene expression patterns .

What are the optimal methods for validating ALDH1A2 antibody specificity?

Establishing antibody specificity is crucial for reliable research results. For ALDH1A2 antibodies, consider:

• Positive and negative controls:

  • Use cells with known ALDH1A2 expression (K-562 cells and mouse testis tissue show positive expression)

  • Include ALDH1A2 knockout or knockdown cells as negative controls

  • Consider species-specific controls when working with cross-reactive antibodies

• Multiple detection techniques:

  • Confirm antibody specificity using Western blot, showing the expected molecular weight of 53-57 kDa or 60 kDa depending on the specific antibody

  • Validate with orthogonal techniques (e.g., mass spectrometry)

  • Use immunoprecipitation followed by Western blot to confirm target specificity

• Functional validation:

  • When studying ALDH1A2 activity, include enzymatic assays for correlation with antibody detection

  • Use ALDH inhibitors like DEAB or disulfiram as functional controls

What are the optimal sample preparation protocols for ALDH1A2 immunohistochemistry?

For successful ALDH1A2 immunohistochemistry experiments:

• Antigen retrieval methods:

  • Primary recommendation: TE buffer pH 9.0

  • Alternative approach: Citrate buffer pH 6.0

• Antibody incubation parameters:

  • For rabbit polyclonal antibodies: Use dilutions ranging from 1:50 to 1:500

  • Optimize incubation time and temperature for specific tissue types

• Validation tissues:

  • Human testis tissue has been validated for positive detection of ALDH1A2

  • Include appropriate negative controls and isotype controls

• Detection systems:

  • Select appropriate secondary antibodies based on the host species of the primary antibody

  • Consider signal amplification methods for low-abundance targets

How can researchers differentiate between ALDH isoforms in their experiments?

ALDH has multiple isoforms with similar structures. To ensure specificity for ALDH1A2:

• Antibody selection:

  • Choose antibodies raised against unique epitopes of ALDH1A2

  • Verify the immunogen information to confirm target specificity

• Expression analysis:

  • TaqMan Low Density Array (TLDA) can be used to screen for expression of different ALDH isoenzymes, as demonstrated in K562 cells

  • RT-PCR with isoform-specific primers can distinguish between ALDH variants

  • RNA-sequencing data can provide comprehensive isoform expression profiles

• Functional distinction:

  • Different ALDH isoforms may have distinct substrate preferences

  • Selective inhibitors can help distinguish between isoform activities

  • Remember that cell lines like K562 express multiple ALDH isoforms including ALDH1, ALDH7, ALDH3, and ALDH8

What are the best practices for preserving ALDH1A2 antibody stability and activity?

To maintain optimal antibody performance:

• Storage conditions:

  • Store ALDH1A2 antibodies at -20°C

  • Antibodies are typically stable for one year after shipment

  • For antibodies in small volumes (e.g., 20μl), aliquoting is unnecessary for -20°C storage

• Buffer composition:

  • ALDH1A2 antibodies are commonly provided in PBS with 0.02% sodium azide and 50% glycerol, pH 7.3

  • Some formulations may contain 0.1% BSA as a stabilizer

• Freeze-thaw considerations:

  • Minimize freeze-thaw cycles

  • For frequent use, consider preparing working aliquots

How can ALDH1A2 be studied in relation to cellular proliferation pathways?

Studies have shown that ALDH1A2 influences cell proliferation:

• Experimental approaches:

  • Overexpression models using lentiviral vectors containing full-length ALDH1A2 cDNA

  • Comparison with appropriate controls (e.g., GFP-expressing cells)

  • Proliferation assays (such as MTT) to quantify growth differences

  • Clonal efficiency assays to assess stem cell-like properties

• Result interpretation:

  • Cells overexpressing ALDH1A2 show higher proliferation rates and clonal efficiency compared to control cells

  • Consider cell type-specific effects, as demonstrated in K562 leukemia and H1299 lung cancer cell lines

• Pathway analysis:

  • Correlate ALDH1A2 expression with known proliferation markers

  • Consider downstream retinoic acid signaling effects

  • Investigate interactions with other genes showing correlated expression patterns

How can ALDH1A2 antibodies contribute to understanding cancer stem cells?

ALDH activity is increasingly recognized as a marker for cancer stem cells:

• Methodology considerations:

  • Use Aldefluor assay in combination with ALDH1A2 antibodies for flow cytometry

  • Sort cells based on ALDH activity (ALDH-high vs. ALDH-low) for further characterization

  • Apply RNA isolation techniques to analyze gene expression differences between populations

• Quality control measures:

  • Verify RNA integrity using methods like the Agilent Bioanalyzer (RNA integrity number)

  • Include appropriate housekeeping genes as endogenous controls (18S, GAPDH, HPRT1, and GUSB have been validated)

• Future research directions:

  • Explore ALDH1A2's role in cancer stem cell maintenance

  • Investigate ALDH1A2 as a potential therapeutic target

  • Study interactions between ALDH1A2 and known cancer stem cell pathways

What are the considerations for multiplexing ALDH1A2 antibodies with other markers?

When designing multiplexed staining protocols:

• Antibody selection criteria:

  • Choose primary antibodies from different host species to avoid cross-reactivity

  • Select fluorophores with minimal spectral overlap for flow cytometry or immunofluorescence

  • Consider sequential staining protocols for challenging combinations

• Controls for multiplexed experiments:

  • Include single-stained controls for compensation

  • Use isotype controls for each antibody

  • Perform blocking steps to minimize non-specific binding

• Data analysis approaches:

  • Use multiparameter analysis software to identify cell populations

  • Consider dimensionality reduction techniques for complex datasets

  • Validate findings with alternative approaches