AKR1B1 Antibody

What is AKR1B1 and what are its primary biological functions?

AKR1B1 (Aldo-Keto Reductase Family 1, Member B1) is a monomeric NADPH-dependent cytosolic enzyme that catalyzes the reduction of a wide variety of carbonyl-containing compounds to their corresponding alcohols. It displays enzymatic activity towards endogenous metabolites such as aromatic and aliphatic aldehydes, ketones, monosaccharides, bile acids, and xenobiotic substrates .

Key biological functions include:

• A central role in the polyol pathway, where it catalyzes the reduction of glucose to sorbitol during hyperglycemia

• Reduction of steroids, their derivatives, and prostaglandins

• Detoxification of dietary and lipid-derived unsaturated carbonyls such as crotonaldehyde, 4-hydroxynonenal, and trans-2-hexenal

• Reduction of phospholipid aldehydes generated from oxidation of phosphatidylcholine and phosphatidylethanolamides

AKR1B1 has been implicated in various pathological conditions, particularly diabetic complications and certain cancers .

What are the standard molecular characteristics of AKR1B1?

How should I validate an AKR1B1 antibody for specificity before use in critical experiments?

Comprehensive validation of AKR1B1 antibodies should include multiple approaches:

• Positive and negative controls: Use tissues or cell lines with known expression levels of AKR1B1. Based on search results, BLBC cell lines (e.g., MDA-MB231, SUM159) show high expression, while luminal breast cancer cell lines (e.g., T47D, MCF7) have low or undetectable expression .

• Knockdown/knockout validation: Compare antibody reactivity in cells with normal versus reduced AKR1B1 expression through siRNA, shRNA, or CRISPR-Cas9 approaches .

• Molecular weight confirmation: Verify that the detected band appears at the expected molecular weight (~34-36 kDa) .

• Cross-reactivity assessment: Test the antibody against recombinant AKR1B1 and related family members (e.g., AKR1B10) to ensure specificity .

• Multiple detection methods: Compare results across different applications (WB, IHC, IF) where possible to confirm consistent detection patterns .

What factors should be considered when choosing between monoclonal and polyclonal AKR1B1 antibodies?

Consider your experimental goals: use monoclonal antibodies when absolute specificity is critical and polyclonal antibodies when maximum sensitivity or cross-species reactivity is needed.

What are the optimal conditions for Western blot detection of AKR1B1?

Sample Preparation:

• Cell/tissue lysis in buffer containing protease inhibitors

• Load 20-40 μg of total protein per lane

• Reduce samples with standard reducing agents (e.g., DTT or β-mercaptoethanol)

Recommended Protocol:

• Separate proteins on 10-12% SDS-PAGE gels

• Transfer to PVDF membrane (recommended over nitrocellulose for AKR1B1)

• Block with 5% BSA (preferred over milk for phospho-specific detection)

• Incubate with primary antibody at recommended dilutions:

  • For monoclonal antibodies: 1:500-1:1000

  • For polyclonal antibodies: 1:500-1:3000

• Wash thoroughly with TBS-T

• Incubate with appropriate HRP-conjugated secondary antibody

• Develop using ECL reagents

Expected Results:

• Single band at approximately 34-36 kDa

• Positive controls: A431 cells, HepG2 cells, human liver tissue, BxPC-3 cells

• Validated antibodies should produce consistent results across these samples

What are the recommended protocols for immunohistochemistry (IHC) and immunofluorescence (IF) using AKR1B1 antibodies?

Immunohistochemistry (IHC):

• Deparaffinize and rehydrate FFPE sections

• Perform heat-induced epitope retrieval (HIER) using citrate buffer (pH 6.0)

• Block endogenous peroxidase with 3% H₂O₂

• Block with 5% normal serum

• Incubate with primary antibody:

  • Recommended dilutions: 1:50-1:200 (antibody-dependent)

  • Incubation time: Overnight at 4°C or 1-2 hours at room temperature

• Apply appropriate HRP-conjugated secondary antibody

• Develop with DAB substrate

• Counterstain, dehydrate, and mount

Immunofluorescence (IF):

• Fix cells with 4% paraformaldehyde

• Permeabilize with 0.1-0.5% Triton X-100

• Block with 5% normal serum

• Incubate with primary antibody:

  • Recommended dilutions: 1:50-1:500

  • Incubation time: Overnight at 4°C or 1-2 hours at room temperature

• Apply fluorophore-conjugated secondary antibody

• Counterstain nuclei with DAPI

• Mount with anti-fade mounting medium

Validated Cell Lines for IF:

• HepG2 cells (consistently show positive staining for AKR1B1)

How can AKR1B1 antibodies be utilized to investigate diabetic complications?

AKR1B1 (aldose reductase) plays a critical role in diabetic complications through the polyol pathway. Research applications include:

• Tissue expression analysis: Use IHC to examine AKR1B1 expression in affected tissues (kidney, retina, peripheral nerves) from diabetic models compared to controls.

• Inhibitor studies: Assess the effects of aldose reductase inhibitors (ARIs) like epalrestat on AKR1B1 expression and activity. Research has shown that epalrestat can suppress the activation of the PKC/NF-κB inflammatory pathway in diabetic complications .

• Mechanistic investigation: Combine AKR1B1 antibodies with markers of the PKC/NF-κB pathway to elucidate signaling mechanisms. The recommended approach includes:

  • Western blot analysis of AKR1B1, PKC-α, and NF-κB p65 expression levels

  • Correlation with inflammatory markers (IL-1β, IL-6, TNF-α)

  • Assessment of tissue damage using H&E staining alongside AKR1B1 immunostaining

• Biomarker development: Quantify AKR1B1 levels in serum or tissue samples using ELISA techniques to evaluate correlation with disease progression or treatment response .

Research has confirmed that inhibiting AKR1B1 effectively suppresses inflammation in sepsis-associated acute kidney injury models, suggesting broader applications beyond diabetic complications .

What is the current understanding of AKR1B1 in cancer research and how can antibodies facilitate these investigations?

AKR1B1 has emerging roles in cancer progression, particularly in basal-like breast cancer (BLBC). Research applications include:

• Expression profiling: AKR1B1 protein expression is significantly elevated in BLBC cell lines (e.g., MDA-MB231, SUM159) but absent in luminal cell lines, making it a potential subtype marker .

• EMT mechanisms: AKR1B1 promotes epithelial-to-mesenchymal transition (EMT) in breast cancer cells. Studies show:

  • Knockdown of AKR1B1 increases E-cadherin expression

  • Inhibition with epalrestat restores E-cadherin expression

  • AKR1B1 represses migration and invasion in vitro

• Signaling pathway analysis: AKR1B1 activates NF-κB signaling through two potential mechanisms:

  • Decrease in NADPH/NADP+ ratio leading to oxidative stress

  • PGF2α-mediated pathway activation

• Transcriptional regulation: Twist2 directly binds to the AKR1B1 promoter at the E-box motif (−997 bp) and activates its transcription in breast cancer cells .

Recommended experimental approaches:

• Use AKR1B1 antibodies for expression analysis across cancer subtypes

• Combine with EMT markers (E-cadherin, vimentin, N-cadherin, Twist2) in multi-label immunofluorescence

• Perform ChIP assays to validate transcription factor binding to the AKR1B1 promoter

• Correlate with clinical outcomes in patient samples

What are common challenges with AKR1B1 antibodies and how can they be addressed?

How can I design a comprehensive experimental approach to investigate AKR1B1's role in cellular redox status?

AKR1B1 influences cellular redox status by affecting the NADPH/NADP+ ratio and reactive oxygen species (ROS) levels. A comprehensive experimental approach should include:

• Expression Analysis:

  • Western blot and immunofluorescence to determine baseline AKR1B1 expression

  • qRT-PCR for mRNA expression

• Functional Manipulation:

  • siRNA/shRNA knockdown of AKR1B1

  • Pharmacological inhibition with epalrestat

  • Ectopic expression in low-expressing cell lines

• Redox Measurements:

  • NADPH/NADP+ ratio quantification

  • ROS levels using fluorescent probes (DCF-DA)

  • Oxidative stress markers (4-HNE, 8-OHdG)

• Downstream Pathway Analysis:

  • NF-κB activation assessment (nuclear translocation, phosphorylation)

  • Co-immunoprecipitation to identify AKR1B1 interaction partners

  • Luciferase reporter assays for NF-κB-dependent transcription

• Integrated Analysis:

  • Correlate AKR1B1 levels with ROS production

  • Assess reversibility of phenotypes with antioxidant treatment

  • Evaluate effects on cell survival, proliferation, and stress response

Research has shown that knockdown of AKR1B1 expression causes a significant decrease in ROS levels, while ectopic AKR1B1 expression induces an increase in ROS . This makes AKR1B1 antibodies essential tools for investigating redox-dependent mechanisms in both normal physiology and disease states.

What are the current challenges in developing isoform-specific antibodies for the AKR1B family?

The AKR1B family includes several members with high sequence homology, making isoform-specific detection challenging. Key considerations include:

• Sequence homology: AKR1B1 shares significant sequence similarity with AKR1B10 (71% amino acid identity), requiring careful epitope selection for antibody development .

• Species conservation: Human AKR1B1 shares approximately 86% amino acid identity with mouse Akr1b3 and rat Akr1b4, complicating cross-species applications .

• Splice variants: Alternative splicing may generate protein variants that are difficult to distinguish with standard antibodies.

• Post-translational modifications: PTMs may affect epitope accessibility or antibody binding.

Recommendations for researchers:

• Target unique regions (C-terminal or N-terminal) for isoform specificity

• Validate with recombinant proteins of all family members

• Perform cross-validation using genetic approaches (knockout/knockdown)

• Consider using multiple antibodies targeting different epitopes for confirmation

How can advanced imaging techniques enhance AKR1B1 localization studies?

While AKR1B1 is primarily described as cytosolic, advanced imaging approaches can reveal nuanced localization patterns relevant to its function:

• Super-resolution microscopy (STORM, PALM, SIM):

  • Achieves resolution below diffraction limit (20-100 nm)

  • Can detect potential membrane associations or organelle-specific pools

  • Requires high-quality, bright fluorophore-conjugated antibodies

• Proximity ligation assay (PLA):

  • Detects protein-protein interactions in situ

  • Useful for studying AKR1B1 interactions with signaling components

  • Combines antibody specificity with signal amplification

• Live-cell imaging with tagged AKR1B1:

  • Complements antibody-based fixed-cell approaches

  • Monitors dynamic localization changes during stress or stimulation

  • Can be validated with antibody staining

• Correlative light and electron microscopy (CLEM):

  • Combines immunofluorescence with ultrastructural information

  • Provides nanometer resolution of AKR1B1 localization

  • Requires specialized sample preparation and antibodies compatible with EM