AKR7A3 Antibody

What is AKR7A3 and what is its biological function?

AKR7A3 belongs to the aldo-keto reductase (AKR) superfamily, whose primary role is to reduce aldehyde substrates to alcohols. The enzyme functions mechanically to reduce the dialdehyde protein-binding form of aflatoxin B1 (AFB1) to the non-binding AFB1 dialcohol . This enzymatic action is believed to protect the liver against the toxic and carcinogenic effects of AFB1, a potent hepatocarcinogen . These enzymes play crucial roles in drug metabolism, carcinogen metabolism, and cellular metabolism .

What types of AKR7A3 antibodies are available for research applications?

Several types of AKR7A3 antibodies are available:

For ELISA-based detection, sandwich ELISA kits are available with detection ranges of 0.78-50 ng/mL (rat) and 0.625-40 ng/mL (human) .

How is AKR7A3 expression altered in hepatocellular carcinoma and what is its significance?

AKR7A3 is frequently down-regulated in hepatocellular carcinoma (HCC) . Transcriptome sequencing of primary HCC samples revealed that AKR7A3 was consistently down-regulated across multiple tumor samples compared to paired non-tumor tissues. In a cohort of 129 HCC patients, qRT-PCR showed significant down-regulation of AKR7A3 (P = 0.0009), with more than 2-fold reduction in 50 out of 129 samples .

The clinical significance of AKR7A3 down-regulation includes:

This data indicates that AKR7A3 may function as a tumor suppressor in HCC .

What functional evidence supports AKR7A3's role as a tumor suppressor?

Multiple functional assays have demonstrated AKR7A3's tumor suppressive properties:

• In vitro assays: Overexpression of AKR7A3 in HCC cell lines (QGY7703 and PLC8024) significantly reduced:

  • Foci formation (P < 0.05 to P < 0.01)

  • Colony formation in soft agar (P < 0.01 to P < 0.001)

  • Cell migration (P < 0.05)

  • Cell invasion (P < 0.05 to P < 0.01)

• In vivo tumor formation: PLC8024-AKR7A3 cells injected subcutaneously into nude mice could not form tumors, while control cells formed tumors in all experimental animals .

• Chemoresistance: AKR7A3 overexpression sensitized HCC cells to cisplatin treatment, demonstrating significantly lower chemoresistance compared to control cells .

What molecular mechanisms underlie AKR7A3's tumor suppressive function?

Western blot analysis revealed that overexpression of AKR7A3 inhibits the activation of several key oncogenic signaling pathways:

• ERK signaling pathway

• c-Jun transcription factor

• NF-κB pathway

These pathways are critical for cancer cell proliferation, survival, and invasion, suggesting that AKR7A3 exerts its tumor suppressive effects by attenuating these pro-oncogenic signaling cascades.

What are the optimal conditions for using AKR7A3 antibodies in Western blot experiments?

Based on validated protocols:

For optimal results:

• Use 12% SDS-PAGE gel for good separation around the 37 kDa region

• Load approximately 30 μg of whole cell lysate per lane

• Include positive controls such as human liver tissue or HepG2 cells, which have detectable AKR7A3 expression

• Include negative controls to verify antibody specificity

How can I design experiments to investigate AKR7A3's role in chemoresistance?

To investigate AKR7A3's role in chemoresistance, consider the following experimental design based on published methodologies:

• Cell line selection: Use HCC cell lines with low endogenous AKR7A3 expression (e.g., QGY7703 and PLC8024)

• Gain-of-function approach:

  • Stably transfect cells with AKR7A3-expressing vector and empty vector control

  • Confirm overexpression by Western blot

  • Treat cells with chemotherapeutic agents (e.g., cisplatin) at various concentrations

  • Measure cell viability using MTT or similar assays

  • Compare IC50 values between AKR7A3-overexpressing and control cells

• Loss-of-function approach:

  • Perform RNA interference (siRNA or shRNA) to knock down AKR7A3 in cells with higher endogenous expression

  • Confirm knockdown efficiency by Western blot

  • Conduct chemosensitivity assays as described above

• Mechanism investigation:

  • Examine activation status of key signaling pathways (ERK, c-Jun, NF-κB) in response to chemotherapy

  • Use Western blot to detect phosphorylated forms of signaling proteins

  • Consider using pathway inhibitors to validate mechanistic findings

What are the best approaches for quantifying AKR7A3 protein levels in clinical samples?

Several validated methods can be employed:

• Western blot:

  • Use validated antibodies (ab227231, 13209-1-AP) at appropriate dilutions

  • Include recombinant AKR7A3 as positive control

  • Normalize to loading controls (β-actin, GAPDH)

  • Use densitometry software for quantification

• ELISA:

  • Commercially available sandwich ELISA kits for human and rat samples

  • Detection range: 0.78-50 ng/mL for rat; 0.625-40 ng/mL for human

  • Minimum detection limit: 0.78 ng/mL for rat kits

  • Suitable for serum, plasma, and tissue homogenates

• Immunohistochemistry (IHC):

  • Validated for paraffin-embedded tissues

  • Recommended antibody dilution: 1:100 for ab227231

  • Score staining intensity and percentage of positive cells

  • Compare with appropriate normal tissue controls

• Mass spectrometry-based approaches:

  • Multiple Reaction Monitoring (MRM) can be used for precise quantification

  • Select unique peptides for AKR7A3 to distinguish from other AKR family members

  • Use recombinant AKR7A3 standards for absolute quantification

Why might I observe multiple bands when using AKR7A3 antibodies in Western blot?

Multiple bands may be observed due to:

• Known molecular weight variations: AKR7A3 has been observed at both 37 kDa (predicted size) and 55-60 kDa in validated Western blots

• Post-translational modifications: Phosphorylation, glycosylation, or other modifications may alter migration patterns

• Cross-reactivity concerns: The high sequence homology among AKR family members can lead to non-specific binding. For example, AKR7A3 shares significant homology with AKR7A2

To address these issues:

• Use recombinant AKR7A3 protein as a positive control to identify the correct band

• Perform knockdown experiments to confirm band specificity

• Consider using multiple antibodies targeting different epitopes of AKR7A3

• Employ antibodies that have been validated for specificity against other AKR family members

How do I distinguish AKR7A3 from other highly homologous AKR family members?

This is a significant challenge due to the high sequence homology among AKR family members. Effective strategies include:

• Select highly specific antibodies:

  • Use antibodies that have been validated against recombinant proteins of multiple AKR family members

  • Confirm no cross-reactions between AKR recombinant proteins using dot blot analysis

• Design specific primers/probes for qRT-PCR:

  • Select primers that amplify unique sequences (100-200 bp)

  • Validate primer specificity by sequencing the amplified products

• For protein detection by mass spectrometry:

  • Select unique peptides for AKR7A3 based on recombinant protein MS/MS data

  • Use data-independent acquisition methods like EMS-EPI scans on mass spectrometers

  • Combine enhanced MS and product ion scans for high-quality signals corresponding to AKR7A3 peptides

• Validate with genetic approaches:

  • Use siRNA or CRISPR to specifically target AKR7A3

  • Verify target knockdown specificity using multiple detection methods

How should I interpret contradictions between mRNA and protein levels of AKR7A3 in my samples?

Discrepancies between mRNA and protein levels are common and may reflect:

• Post-transcriptional regulation: miRNAs or RNA-binding proteins may affect translation efficiency

• Protein stability differences: AKR7A3 protein may have different turnover rates in different tissues or conditions

• Technical considerations:

  • mRNA detection is typically more sensitive than protein detection

  • Antibody affinity and specificity issues may affect protein quantification

To address these discrepancies:

• Use multiple detection methods for both mRNA (qRT-PCR, RNA-seq) and protein (Western blot, ELISA, IHC)

• Consider pulse-chase experiments to examine protein stability

• Investigate potential regulatory mechanisms specific to your experimental context

• Always include appropriate positive and negative controls in both mRNA and protein assays

What are promising areas for future research using AKR7A3 antibodies?

Several promising research directions emerge from current findings:

• Cancer biomarker development:

  • The significant association between AKR7A3 down-regulation and poor prognosis in HCC suggests potential utility as a prognostic biomarker

  • Development of sensitive and specific detection methods for clinical samples could enable translation to diagnostic applications

• Therapeutic targeting:

  • Given its tumor suppressive role, strategies to restore AKR7A3 expression or activity could be explored

  • Drug development targeting pathways regulated by AKR7A3 (ERK, c-Jun, NF-κB) may be beneficial in cancers with AKR7A3 down-regulation

• Detoxification mechanisms:

  • Further characterization of AKR7A3's role in detoxifying environmental carcinogens beyond aflatoxin B1

  • Investigation of potential chemopreventive strategies based on AKR7A3 induction

• Advanced antibody applications:

  • Development of antibodies suitable for chromatin immunoprecipitation (ChIP) to study AKR7A3 interactions with chromatin

  • Generation of antibodies specific for post-translationally modified forms of AKR7A3 to better understand its regulation

What technological advances might improve AKR7A3 antibody specificity and utility?

Emerging technologies offer opportunities to enhance AKR7A3 antibody applications:

• Single-cell protein analysis:

  • Adaptation of AKR7A3 antibodies for mass cytometry (CyTOF) or imaging mass cytometry

  • Development of highly specific antibodies for single-cell Western blot applications

• Proximity-based assays:

  • Utilization of AKR7A3 antibodies in proximity ligation assays (PLA) to study protein-protein interactions

  • Implementation in BRET/FRET systems to investigate real-time interactions in living cells

• Nanobody development:

  • Engineering of small, high-affinity nanobodies against AKR7A3 for improved tissue penetration and reduced background

  • Development of bispecific antibodies targeting AKR7A3 and its interaction partners

• Epitope mapping technologies:

  • Detailed characterization of epitope specificity using hydrogen/deuterium exchange mass spectrometry or cryo-EM

  • Rational design of antibodies targeting functional domains of AKR7A3