ALDH22A1 Antibody

What are the validated applications for ALDH1A2 antibody?

ALDH1A2 antibody (such as 13951-1-AP) has been extensively validated for multiple applications including Western Blot (WB), Immunohistochemistry (IHC), Immunoprecipitation (IP), and ELISA. The antibody has demonstrated positive Western Blot detection in K-562 cells and mouse testis tissue, while Immunoprecipitation has been validated in mouse testis tissue. Immunohistochemistry applications have been confirmed in human testis tissue . Published literature supports these applications, with at least 9 publications documenting WB applications and 5 publications supporting IHC applications .

What are the recommended dilution ratios for different experimental procedures?

For optimal results with ALDH1A2 antibody, the following dilution ranges are recommended:

It is important to note that optimal dilutions are sample-dependent and should be determined experimentally for each testing system . Researchers should perform a titration series when working with new sample types to determine the optimal signal-to-noise ratio.

What antigen retrieval methods are most effective for ALDH1A2 immunohistochemistry?

For ALDH1A2 antibody in IHC applications, the suggested antigen retrieval method is TE buffer at pH 9.0. Alternatively, antigen retrieval may be performed with citrate buffer at pH 6.0 . When working with formaldehyde-fixed, paraffin-embedded tissue sections, complete deparaffinization and rehydration are essential prerequisites before antigen retrieval . This is particularly important as improper antigen retrieval can significantly impact antibody binding efficiency and result in false negative or high background staining.

What are the optimal storage conditions for preserving ALDH antibody activity?

ALDH1A2 antibody should be stored at -20°C in PBS buffer containing 0.02% sodium azide and 50% glycerol at pH 7.3. Under these conditions, the antibody remains stable for one year after shipment . Notably, aliquoting is unnecessary for -20°C storage, which simplifies laboratory workflow. For preparations containing 20μl, it's important to note that they contain 0.1% BSA as a stabilizer . Avoiding repeated freeze-thaw cycles is advisable to maintain antibody performance over time.

How can researchers investigate ALDH1A2's role in embryonic development?

ALDH1A2, also known as RALDH2 (retinal dehydrogenase 2), exhibits complex expression patterns throughout embryonic development as a cytosolic homotetramer comprising 56.7 kDa subunits . To investigate its developmental role, researchers should:

• Utilize developmental stage-specific tissue sampling with precise temporal controls

• Employ immunofluorescence co-staining with developmental markers to characterize expression patterns

• Consider conditional knockout models to investigate stage-specific functions

• Use the validated antibody dilution of 1:50-1:500 for IHC in embryonic tissues

• Implement comparative analysis between ALDH1A2 and other ALDH family members to distinguish isoform-specific roles

The antibody's validated reactivity in human, mouse, and rat samples makes it valuable for cross-species developmental studies, enabling evolutionary insights into ALDH1A2 function .

What role does ALDH1A3 play in cellular metabolism and how can antibodies help investigate this?

ALDH1A3 serves as a critical metabolic coordinator that links metabolism with gene regulation. Research shows that ALDH1A3 is upregulated in pulmonary arterial hypertension (PAH) versus control pulmonary artery smooth muscle cells (PASMC) . To investigate ALDH1A3's metabolic functions:

• Use immunofluorescence with ALDH1A3-specific antibodies (1:100 dilution, Abgent) on tissue sections to assess protein localization and expression levels

• Implement siRNA knockdown experiments to evaluate ALDH1A3's impact on proliferation and glycolysis

• Employ metabolic assays such as extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) measurements following ALDH1A3 manipulation

• Examine nuclear ALDH1A3's role in converting acetaldehyde to acetate to produce acetyl-CoA for histone acetylation

Studies have demonstrated that ALDH1A3 knockdown reduces proliferation in PAH PASMC and decreases glycolysis as measured by ECAR, highlighting its importance in cellular energetics and proliferative capacity .

How can researchers differentiate between ALDH family members in experimental designs?

Given the 19 described ALDH isoenzymes , distinguishing between family members requires careful experimental design:

• Select antibodies with validated specificity for the target ALDH isoform

• Confirm antibody specificity through protein arrays, as exemplified by Prestige Antibodies testing against 364 human recombinant protein fragments

• Include genetic knockdown controls to verify antibody specificity

• Use precise molecular weight determination (ALDH1A2 is observed at 53-57 kDa compared to its calculated 57 kDa)

• Consider transcriptomic analysis to identify differentially expressed ALDH isoforms before selecting antibody targets

For example, in PAH research, transcriptomic analysis identified ALDH1A3 as the only significantly changed ALDH isoenzyme among the 19 family members, enabling focused investigation with specific antibodies .

What factors contribute to variability in ALDH detection across different tissue types?

Several factors can influence ALDH detection variability across tissues:

• Differential expression levels: ALDH1A2 demonstrates tissue-specific expression patterns, with confirmed detection in K-562 cells and testis tissue

• Fixation protocols: Formaldehyde fixation can mask epitopes differently across tissue types, necessitating optimization of antigen retrieval methods

• Buffer composition: Antigen retrieval with TE buffer (pH 9.0) versus citrate buffer (pH 6.0) can dramatically affect detection sensitivity

• Endogenous enzyme activity: Tissues with high endogenous peroxidase activity may require additional blocking steps

• Tissue-specific interfering substances: Lipid content, pigmentation, and autofluorescence can interfere with antibody binding and signal detection

To address these variables, researchers should include positive control tissues (such as testis for ALDH1A2) and perform parallel staining with multiple antibody dilutions to establish optimal protocols for each tissue type .

How should researchers interpret discrepancies between protein expression levels detected by different ALDH antibodies?

When confronted with discrepant results between different ALDH antibodies:

• Compare antibody epitopes: Different antibodies may target distinct regions of the same protein, affecting detection sensitivity

• Evaluate antibody validation data: Compare Western blot bands with expected molecular weights (ALDH1A2 should appear at 53-57 kDa)

• Assess isoform specificity: Consider whether antibodies might cross-react with related ALDH family members

• Review sample preparation methods: Differences in antigen retrieval or protein extraction protocols can affect epitope availability

• Implement orthogonal validation: Confirm protein expression using complementary techniques such as mass spectrometry or mRNA quantification

For instance, ALDH1A2 antibody (13951-1-AP) has been validated for specificity with observed molecular weight of 53-57 kDa, providing a reference point for evaluating potential discrepancies .

What experimental controls are essential when investigating ALDH's role in disease models?

When studying ALDH proteins in disease models, such as pulmonary arterial hypertension or ischemic conditions, include these controls:

• Appropriate tissue/cell controls: Compare disease samples with matched healthy controls (as demonstrated in PAH vs. control PASMC studies)

• Genetic manipulation controls: Include siRNA knockdown validation (as used in ALDH1A3 studies) to confirm antibody specificity

• Isotype controls: Implement matching isotype antibodies to assess non-specific binding

• Positive tissue controls: Include known positive tissues such as testis for ALDH1A2

• Loading controls: For Western blots, use established housekeeping proteins appropriate for the tissue/condition

In PAH research, ALDH1A3 knockdown experiments included verification of efficient knockdown and equal cell density controls for metabolic assays, providing confidence in observed phenotypic changes .

How can researchers design experiments to investigate interactions between ALDH activity and oxidative stress?

To study ALDH's relationship with oxidative stress:

• Measure complementary markers: Assess superoxide dismutase (SOD) activity and malondialdehyde (MDA) content alongside ALDH expression

• Implement pharmacological interventions: Test ALDH activators like Alda-1 (N-(1,3-benzodioxol-5-ylmethyl)-2,6-dichlorobenzamide) to evaluate protective effects

• Evaluate downstream pathway components: Examine mitophagy-related proteins (PINK1, Parkin, Beclin-1, p62, LC3) to understand mechanistic connections

• Include inflammatory markers: Measure cytokines like IL-1β, IL-6, and TNF-α that may link ALDH activity to inflammatory processes

• Assess functional outcomes: Connect molecular changes to functional improvements, such as tissue survival or vascular density

Research has shown that ALDH2 activation by Alda-1 enhanced skin flap survival by reducing oxidative stress, demonstrating increased SOD activity and decreased MDA levels, while also inhibiting mitophagy through the PINK1/Parkin pathway .

What considerations are important when using ALDH antibodies in multi-color immunofluorescence studies?

For successful multi-color immunofluorescence with ALDH antibodies:

• Carefully select compatible primary antibody hosts: For ALDH1A2 (rabbit host) , pair with antibodies from different species

• Optimize antibody dilutions: Start with recommended ranges (1:50-1:500 for ALDH1A2 IHC) but perform titration series for each application

• Plan appropriate fluorophore combinations: Consider spectral overlap and use sequential imaging when necessary

• Implement proper controls: Include single-stained samples and fluorophore minus one (FMO) controls

• Validate co-localization: Confirm expected subcellular localization patterns (cytosolic for ALDH1A2, potentially nuclear for ALDH1A3)

Successful multi-color immunofluorescence has been demonstrated in studies co-staining for ALDH1A3 with SM22α, PCNA, PKM2, NFYA, or active β-catenin, enabling detailed analysis of ALDH1A3's relationship with proliferation and metabolic markers .

How might advances in antibody technology enhance ALDH research in the coming years?

Future ALDH antibody research may benefit from these emerging technologies:

• Single-cell protein analysis: Developing antibodies compatible with mass cytometry or similar technologies for single-cell resolution of ALDH expression

• Live-cell imaging compatible antibodies: Creating non-toxic cell-permeable antibody fragments for dynamic ALDH tracking

• Super-resolution microscopy: Optimizing antibodies for techniques like STORM or PALM to resolve subcellular localization with nanometer precision

• Proximity labeling approaches: Combining ALDH antibodies with proximity labeling technologies to identify novel interaction partners

• Conformation-specific antibodies: Developing antibodies that distinguish between active and inactive ALDH conformations

These approaches would allow researchers to move beyond static measurements of ALDH expression toward understanding dynamic regulation and function in living systems.

What novel research applications might emerge from combining ALDH antibodies with emerging analytical techniques?

Integrating ALDH antibodies with cutting-edge analytical methods could enable:

• Spatial transcriptomics correlation: Aligning antibody-based protein localization with spatial mRNA expression data

• Single-cell multi-omics: Combining ALDH protein detection with transcriptomic and metabolomic profiling at single-cell resolution

• Artificial intelligence-assisted image analysis: Developing deep learning models to quantify subtle changes in ALDH expression patterns across tissues

• Organoid and tissue chip applications: Utilizing ALDH antibodies to study dynamic protein expression in 3D culture systems

• In vivo imaging probes: Developing antibody-based tracers for non-invasive monitoring of ALDH activity in animal models

These integrative approaches would provide unprecedented insights into ALDH biology across scales from molecular interactions to whole-organism physiology.