AKR7A3 Antibody

What is AKR7A3 and why is it significant in cancer research?

Methodologically, when investigating AKR7A3's role in cancer research, consider:

• Analyzing expression patterns across tumor and normal tissues

• Correlating expression levels with clinical parameters

• Examining its functional interaction with known oncogenic pathways

What detection methods are available for AKR7A3 in experimental settings?

Multiple detection methods have been validated for AKR7A3:

• ELISA: Sandwich ELISA methods can detect AKR7A3 in various sample types with a detection range of 0.78-50 ng/mL and a minimum detection limit of 0.78 ng/mL .

• Western Blot: Successfully used to analyze AKR7A3 protein levels in both tissue samples and cell lines .

• qRT-PCR: Effective for measuring AKR7A3 mRNA expression levels in research samples, as demonstrated in studies with 129 pairs of HCC patient samples .

When selecting a detection method, consider your sample type (tissue homogenate, serum, cell lysate), required sensitivity, and whether you need quantitative or qualitative results.

What sample types can be used for AKR7A3 detection?

AKR7A3 can be detected in multiple biological sample types:

• Serum

• Plasma

• Urine

• Tissue homogenates

• Cell culture supernatants

• Other biological fluids

For optimal results when working with tissue samples, proper homogenization and extraction protocols should be followed to preserve protein integrity.

How does AKR7A3 expression correlate with hepatocellular carcinoma progression and clinical outcomes?

Research has established significant correlations between AKR7A3 down-regulation and several clinical parameters:

Methodologically, researchers should employ multivariate analysis when examining such correlations to account for confounding factors.

What signaling pathways are modulated by AKR7A3 in cancer cells?

AKR7A3 influences several critical signaling pathways in cancer:

• MAPK/ERK Pathway: AKR7A3 inhibits ERK phosphorylation, which is significant because ERK1/2 are constitutively activated in various tumors and promote cell proliferation and metastasis .

• c-Jun Pathway: Cells overexpressing AKR7A3 demonstrate reduced c-Jun activity. This is noteworthy as c-Jun protects hepatocytes from apoptosis by antagonizing p53 functions .

• NF-κB Pathway: AKR7A3 attenuates NF-κB activity, which typically activates anti-apoptotic proteins leading to uncontrolled cell proliferation .

When investigating these pathways, western blot analysis targeting phosphorylated forms of these signaling molecules provides reliable quantitative data on pathway activation status.

How does AKR7A3 influence chemoresistance in HCC?

Experimental data demonstrates that AKR7A3 overexpression significantly reduces chemoresistance in HCC cells treated with cisplatin . This suggests AKR7A3 sensitizes cancer cells to chemotherapy.

Methodological approach for studying this phenomenon:

• Establish stable cell lines with AKR7A3 overexpression or knockdown

• Treat cells with varying concentrations of chemotherapeutic agents

• Assess cell viability using standardized assays (MTT, CCK-8)

• Analyze apoptotic markers and pathways via western blot and flow cytometry

• Compare IC50 values between AKR7A3-modified and control cells

What mechanisms contribute to AKR7A3 down-regulation in HCC?

Two primary mechanisms have been identified:

• Promoter Hypermethylation: The promoter region of AKR7A3 is frequently hypermethylated in HCC, leading to transcriptional silencing .

• Loss of Heterozygosity (LOH): Chromosomal deletions affecting the AKR7A3 locus have been detected in HCC samples .

For investigating these mechanisms, researchers should employ:

• Bisulfite sequencing or methylation-specific PCR for methylation analysis

• SNP analysis for LOH detection (primers flanking SNP positions rs1738025 and rs2231198 have been validated)

What functional assays are recommended for studying AKR7A3's tumor suppressive properties?

Based on published research, these assays have proven effective:

• In vitro assays:

  • Foci formation assay: Measures anchorage-dependent growth and contact inhibition

  • Soft agar colony formation: Assesses anchorage-independent growth (hallmark of transformation)

  • Migration assay: Evaluates cellular motility

  • Invasion assay: Measures ability to penetrate extracellular matrix

• In vivo assays:

  • Subcutaneous tumor formation in nude mice: PLC8024-AKR7A3 overexpressing cells showed complete inhibition of tumor formation compared to control cells

When designing these experiments, include both gain-of-function (overexpression) and loss-of-function (knockdown) approaches for comprehensive analysis.

What are best practices for validating AKR7A3 antibody specificity?

Although the search results don't specifically address AKR7A3 antibody validation, standard methodological approaches include:

• Western blot analysis comparing:

  • Positive and negative control samples

  • AKR7A3 knockdown vs. control cells

  • Recombinant AKR7A3 protein dilutions

• Immunoprecipitation followed by mass spectrometry to confirm target identity

• Immunohistochemistry controls:

  • Omitting primary antibody

  • Blocking peptide competition

  • Testing on tissues with known AKR7A3 expression profiles

What protocol considerations are important when using ELISA for AKR7A3 detection?

When using sandwich ELISA for AKR7A3 detection:

• Sample preparation:

  • Do not mix reagents from different kit lots

  • Calculate adequate sample volumes before starting

  • Dilute samples that generate values exceeding the highest standard

• Assay procedure:

  • Pre-coated plates with an antibody specific to AFB1-AR are used

  • Samples are added with a biotin-conjugated polyclonal antibody preparation specific for AFB1-AR

  • Avidin conjugated to Horseradish Peroxidase (HRP) is added and incubated

  • TMB substrate solution addition results in color change

  • The reaction is terminated with sulfuric acid solution

  • Measure absorbance at 450 nm ± 2 nm

• Result analysis:

  • Determine concentration by comparing sample O.D. to standard curve

  • Expected detection range: 0.78-50 ng/mL

  • Minimum detection limit: 0.78 ng/mL

How should contradictory results in AKR7A3 functional studies be interpreted?

When encountering contradictory results:

• Methodological assessment:

  • Verify antibody specificity using multiple validation techniques

  • Confirm target knockdown/overexpression efficiency at both mRNA and protein levels

  • Assess potential off-target effects using multiple siRNA/shRNA constructs

• Biological context evaluation:

  • Cell type-specific effects may occur (compare results across multiple cell lines)

  • Consider microenvironmental factors that may affect AKR7A3 function

  • Examine potential compensatory mechanisms through pathway analysis

• Experimental design review:

  • Ensure appropriate positive and negative controls were included

  • Verify statistical analysis and sample sizes are adequate

  • Consider time-dependent effects in your experimental system

What factors affect reproducibility when studying AKR7A3 in experimental systems?

Several factors can impact reproducibility:

• Experimental variables:

  • Operator variation in pipetting technique

  • Washing technique variations

  • Incubation time or temperature fluctuations

  • Kit/reagent age and storage conditions

• Biological variables:

  • Cell passage number and culture conditions

  • Sample collection and processing protocols

  • Patient heterogeneity in clinical samples

• Technical considerations:

  • Antibody lot-to-lot variation

  • Cross-reactivity with related proteins

  • Assay sensitivity limitations

To improve reproducibility, standardize protocols, use consistent reagent sources, and implement rigorous quality control measures.

How can AKR7A3's tumor suppressive functions be reconciled with its enzymatic activity?

While AKR7A3 is known as an aflatoxin aldehyde reductase, its tumor suppressive functions appear to extend beyond this enzymatic activity. Research indicates AKR7A3 inhibits multiple oncogenic signaling pathways:

• Dual functionality hypothesis: Consider that AKR7A3 may have both enzymatic functions (detoxification) and signaling functions (pathway inhibition)

• Methodological approach to investigation:

  • Compare wild-type and catalytically inactive mutants in functional assays

  • Assess correlation between enzymatic activity and tumor suppression

  • Identify protein-protein interactions that might mediate non-enzymatic functions

• Data interpretation framework:

  • Determine if tumor suppression is dependent on or independent of enzymatic activity

  • Consider potential metabolic products that might influence signaling pathways

  • Examine expression patterns of both AKR7A3 and its substrates in cancer tissues

What are promising areas for future AKR7A3 antibody applications in cancer research?

Several directions warrant further investigation:

• Biomarker development:

  • Given the correlation between AKR7A3 down-regulation and poor prognosis, developing standardized immunohistochemical protocols for clinical use

  • Exploring liquid biopsy applications to detect circulating AKR7A3

• Therapeutic targeting:

  • Investigating compounds that can restore AKR7A3 expression in cancer cells

  • Exploring combination therapies that leverage AKR7A3's ability to reduce chemoresistance

• Mechanistic studies:

  • Further characterizing the molecular interactions between AKR7A3 and ERK, c-Jun, and NF-κB pathways

  • Investigating potential post-translational modifications that regulate AKR7A3 activity

How might AKR7A3 expression patterns inform personalized medicine approaches?

Research suggests AKR7A3 expression correlates with both clinical outcomes and treatment response:

• Stratification strategy development:

  • Generate cohort-based cutoff values for "low" versus "high" AKR7A3 expression

  • Correlate expression levels with response to specific therapeutic regimens

• Methodological considerations:

  • Standardize quantification methods across clinical laboratories

  • Develop and validate companion diagnostic assays

  • Integrate AKR7A3 status with other molecular markers for comprehensive profiling

• Clinical application framework:

  • Monitor AKR7A3 expression before and during treatment to detect changes

  • Use expression patterns to guide chemotherapy selection based on demonstrated sensitization effects