AKR1C1 Antibody

What is AKR1C1 and why is it important in research?

AKR1C1 (aldo-keto reductase family 1 member C1) is a member of the aldo/keto reductase protein family that catalyzes NAD(P)H-dependent reduction of carbonyl groups in various substrates. It is a cytoplasmic protein with a reported length of 323 amino acid residues and a molecular mass of approximately 36.8 kDa . The protein is widely expressed in human tissues including liver, prostate, testis, adrenal gland, brain, uterus, mammary gland, and keratinocytes . AKR1C1 plays critical roles in cholesterol metabolism and homeostasis, as well as epithelial cell differentiation . It has also been implicated in various pathological conditions, particularly in cancer progression, making it a significant target for research in oncology and metabolic disorders .

The protein is also known by several synonyms including 20-ALPHA-HSD, C9, DD1, DDH, DDH1, H-37, and 2-ALPHA-HSD, which reflects its diverse functional activities in different biological contexts . Understanding AKR1C1's functions and regulatory mechanisms requires specific antibodies that can accurately detect and quantify this protein in various experimental settings.

What types of AKR1C1 antibodies are available for research applications?

Several types of AKR1C1 antibodies are available for research applications, including monoclonal and polyclonal antibodies. Monoclonal antibodies, such as clone 859026 from R&D Systems, offer high specificity and consistent performance across different experimental batches . These antibodies are typically generated against recombinant human AKR1C1 protein that encompasses the full-length sequence (Met1-Tyr323) .

Antibodies against AKR1C1 are available with different host species, conjugations, and applications. When selecting an antibody, researchers should consider the specific experimental requirements, including the detection method (Western blot, immunohistochemistry, ELISA, etc.), sample type (cell lysate, tissue section, etc.), and the degree of specificity required . Validation information, such as positive and negative control tissues or cell lines, should be examined to ensure antibody performance in the intended application.

How can I optimize Western blot protocols for AKR1C1 detection?

For optimal Western blot detection of AKR1C1, researchers should consider several critical factors. Based on published protocols, the following optimization guidelines are recommended:

• Sample preparation: Total protein extraction from cells or tissues should be performed with appropriate lysis buffers containing protease inhibitors to prevent degradation. For tissue samples, proper homogenization is essential .

• Protein loading and separation: Load 20-50 μg of total protein per lane. AKR1C1 has an expected band size of approximately 37 kDa, though it may appear at approximately 41 kDa in some gel systems . Use 10-12% polyacrylamide gels for optimal separation.

• Transfer conditions: Transfer to PVDF membranes is typically performed using standard wet transfer systems.

• Antibody dilution: Primary antibody dilutions vary by product; for instance, Mouse Anti-Human AKR1C1 Monoclonal Antibody (MAB6529) has been successfully used at 0.1 μg/mL, while some other antibodies like GeneTex GTX105620 have been used at dilutions of 1:5000 .

• Incubation conditions: Primary antibody incubation is typically performed overnight at 4°C, followed by appropriate HRP-conjugated secondary antibody incubation (e.g., HAF007) at room temperature for 1 hour .

• Detection system: ECL detection reagents are commonly used, with imaging performed on systems such as Amersham Imager 600 or GelView 6000M .

For validation purposes, HepG2 human hepatocellular carcinoma cell line and human liver tissue are commonly used as positive controls for AKR1C1 expression .

What are the best practices for immunohistochemical detection of AKR1C1?

For immunohistochemical (IHC) detection of AKR1C1 in tissue samples, researchers should follow these methodological guidelines:

• Tissue preparation: Paraffin-embedded tissues should be properly dewaxed and rehydrated before antigen retrieval .

• Antigen retrieval: Heat-induced epitope retrieval using sodium citrate buffer (pH 6.0) for 30 minutes in a microwave oven has been successfully employed for AKR1C1 detection .

• Blocking and antibody incubation: Follow manufacturer's protocols for blocking non-specific binding sites. For primary antibody incubation, AKR1C1 antibody (e.g., GTX105620, GeneTex) has been successfully used at a dilution of 1:2000 .

• Detection system: Streptavidin-peroxidase (SP) kits such as KIT 9710 (MAIXIN) have been used according to manufacturer's protocols .

• Imaging and quantification: Images can be captured using an inverted microscope (e.g., Leica DMI 4000 B) and analyzed using image analysis software such as Image-Pro Plus for quantitative assessment .

When conducting IHC studies, appropriate positive and negative controls should be included. Normal lung tissue samples have been used as controls in comparison to cancerous lung tissues for AKR1C1 expression studies .

How does AKR1C1 contribute to non-small cell lung cancer (NSCLC) progression?

AKR1C1 has been implicated in NSCLC progression through several mechanisms:

• Metabolic reprogramming: Recent research has demonstrated that AKR1C1 promotes NSCLC cell proliferation by reprogramming tumor metabolism . AKR1C1 has been shown to augment the expression of hypoxia-inducible factor 1-alpha (HIF-1α), which drives tumor metabolic reprogramming to support cancer cell growth .

• Lactate production: Studies have shown that AKR1C1 knockdown decreases lactate production in NSCLC cells, suggesting a role in glycolytic metabolism which is a hallmark of cancer cells .

• HIF-1α pathway modulation: AKR1C1 modulates HIF-1α protein levels and the expression of its target genes in NSCLC cells, establishing a mechanistic link between AKR1C1 and metabolic adaptation in cancer .

• Clinical correlation: High expression of AKR1C1 significantly correlates with HIF-1α signaling and predicts poor prognosis for NSCLC patients, highlighting its potential value as a prognostic biomarker .

Researchers investigating AKR1C1 in cancer should consider examining these metabolic pathways and incorporate appropriate metabolic assays, such as lactate measurements, alongside protein expression studies to fully understand AKR1C1's role in cancer progression.

What techniques are recommended for studying AKR1C1's role in chemoresistance?

To investigate AKR1C1's role in chemoresistance, researchers should consider the following methodological approaches:

• Expression modulation studies: Utilize both loss-of-function (siRNA, shRNA) and gain-of-function (overexpression) approaches to manipulate AKR1C1 levels in cancer cell lines. Stable AKR1C1 silencing and overexpression can be achieved through lentivirus-delivered technology, as described in published protocols .

• Drug sensitivity assays: After modulating AKR1C1 expression, assess cellular sensitivity to chemotherapeutic agents using viability assays (MTT, SRB) or apoptosis assays (Annexin V/PI staining, caspase activation) .

• Metabolic assessments: Since AKR1C1 is involved in metabolic reprogramming, include measurements of key metabolic parameters such as lactate production. This can be done using colorimetric or fluorometric assay kits (e.g., Biovision K607-100) following the manufacturer's protocols .

• Molecular pathway analysis: Examine the effect of AKR1C1 modulation on drug resistance-related pathways, particularly focusing on HIF-1α and its downstream targets, using Western blot and qRT-PCR techniques .

• In vivo models: Consider using xenograft models with AKR1C1-modulated cancer cells to assess therapeutic responses in a more physiologically relevant context.

Research has shown that the reductase activity of AKR1C1 contributes to resistance against various chemotherapeutic agents, including cisplatin and daunorubicin in different cancer types . The connection between AKR1C1-mediated metabolic alterations and drug resistance mechanisms presents important avenues for therapeutic intervention.

How can researchers distinguish between AKR1C1 and other highly homologous AKR1C family members?

Distinguishing between AKR1C1 and other highly homologous family members (AKR1C2, AKR1C3, and AKR1C4) presents a significant challenge in research due to their high sequence similarity. Researchers should consider the following approaches:

• Antibody selection: Choose antibodies specifically validated for their lack of cross-reactivity with other AKR1C family members. Validation data should explicitly demonstrate specificity through techniques such as testing on tissues known to differentially express AKR1C isoforms .

• RNA interference specificity: When designing siRNA or shRNA for knockdown experiments, carefully select sequences unique to AKR1C1. Testing the effect of knockdown on all family members using isoform-specific qPCR is recommended to confirm specificity .

• Expression pattern analysis: Leverage the differential expression patterns of AKR1C family members across tissues. While AKR1C1, AKR1C2, and AKR1C3 have broad expression profiles, AKR1C4 is predominantly expressed in the liver . This can help validate findings in certain tissue contexts.

• Functional assays: Utilize the distinct substrate preferences and enzymatic efficiencies of different AKR1C enzymes. AKR1C1 preferentially catalyzes the reduction of specific 20-ketosteroids, which can be exploited in biochemical assays to distinguish its activity .

• Mass spectrometry-based approaches: For definitive identification, consider proteomics approaches that can detect isoform-specific peptides, particularly from regions where amino acid sequences differ between family members.

Each of these approaches has limitations, and researchers should ideally use multiple methods in combination to ensure accurate identification of AKR1C1 in their experimental systems.

What are the recommended protocols for analyzing AKR1C1's involvement in metabolic reprogramming?

To investigate AKR1C1's role in metabolic reprogramming, particularly in cancer contexts, researchers should implement a multi-faceted approach:

• Lactate production assays: Measure extracellular lactate levels in culture medium from cells with modulated AKR1C1 expression. For accurate results, seed cells at consistent densities (e.g., 4×10³ cells/well in 96-well plates), transfect with siRNA/control RNA, and collect medium after standardized incubation periods. Analyze using colorimetric/fluorometric assay kits with absorbance measured at 570 nm .

• Glycolytic flux analysis: Employ extracellular flux analyzers (e.g., Seahorse XF) to measure glycolytic rate and capacity in real-time. This provides dynamic information about the impact of AKR1C1 on cellular energy metabolism.

• HIF-1α pathway analysis: Given AKR1C1's connection to HIF-1α, assess both HIF-1α protein levels and the expression of its downstream targets. Use Western blot with appropriate antibodies (e.g., 1:1000, 610958, BD Biosciences for HIF-1α) and qRT-PCR for target gene expression analysis .

• Metabolomics approaches: Consider untargeted metabolomics to identify broader metabolic alterations associated with AKR1C1 modulation. This can reveal unexpected metabolic pathways affected by AKR1C1 activity.

• Isotope tracing: Utilize stable isotope-labeled substrates (e.g., ¹³C-glucose or ¹³C-glutamine) followed by mass spectrometry analysis to track specific metabolic pathways and determine how AKR1C1 affects substrate utilization and metabolic flux.

When interpreting results, it's important to normalize metabolic measurements to cell number or protein content to account for potential effects of AKR1C1 on cell proliferation that could confound metabolic data .

How can researchers validate the specificity of AKR1C1 antibodies?

Validating antibody specificity is crucial for obtaining reliable results in AKR1C1 research. Researchers should implement the following validation strategies:

• Positive and negative control samples: Test antibodies on tissues or cell lines known to express or lack AKR1C1. HepG2 human hepatocellular carcinoma cell line and human liver tissue are commonly used as positive controls for AKR1C1 expression .

• Knockdown/knockout controls: Validate antibody specificity by comparing signal between wild-type samples and those with AKR1C1 knockdown or knockout. This approach provides a stringent test of antibody specificity, particularly important for distinguishing between highly homologous family members .

• Recombinant protein controls: Use purified recombinant AKR1C1 protein as a positive control in Western blots, along with other recombinant AKR1C family members to test for cross-reactivity .

• Multiple antibody comparison: When possible, use multiple antibodies targeting different epitopes of AKR1C1 to corroborate findings.

• Multiple detection methods: Validate expression using complementary techniques (e.g., if performing IHC, confirm with Western blot or qPCR) to strengthen confidence in antibody specificity .

• Expected molecular weight verification: Confirm that the detected protein band appears at the expected molecular weight (~37 kDa for AKR1C1, though it may appear at ~41 kDa in some gel systems) .

Antibody manufacturers typically provide validation data, but researchers should perform their own validation in the specific experimental system they are using, as antibody performance can vary depending on sample preparation, detection methods, and experimental conditions .

What are common pitfalls in AKR1C1 experimental designs and how can they be addressed?

Researchers studying AKR1C1 should be aware of several common pitfalls and implement appropriate strategies to address them:

• Family member cross-reactivity: The high sequence homology between AKR1C family members (AKR1C1-C4) makes cross-reactivity a significant concern. To address this:

  • Use validated isoform-specific antibodies

  • Confirm specificity through knockdown/knockout approaches

  • Consider complementary RNA-based detection methods for validation

• Post-translational modifications: AKR1C1 may undergo various post-translational modifications that can affect antibody recognition or protein function. Researchers should:

  • Consider using phospho-specific antibodies if studying regulatory mechanisms

  • Be aware that modifications might alter protein migration in gels

  • Optimize sample preparation to preserve relevant modifications

• HIF-1α stability issues: When studying AKR1C1's relationship with HIF-1α, remember that HIF-1α is highly unstable under normoxic conditions. To address this:

  • Process samples quickly and maintain cold temperatures

  • Divide samples into several tubes and store at -80°C to avoid freeze-thaw cycles

  • Use hypoxia-mimetic compounds if appropriate for the research question

• Metabolic assay confounding factors: When conducting metabolic studies:

  • Normalize metabolic measurements to cell number or protein content

  • Control for differences in cell proliferation rates

  • Standardize culture conditions (confluence, medium composition, incubation time)

• Functional redundancy: AKR1C family members may have partially overlapping functions, complicating interpretation of single gene modulation experiments. Consider:

  • Measuring activity of multiple family members simultaneously

  • Using combination knockdowns to address functional redundancy

  • Employing enzyme activity assays alongside expression analyses

By anticipating these common pitfalls and implementing appropriate experimental controls, researchers can enhance the reliability and reproducibility of their AKR1C1 studies.