DHRS2 Antibody

What is DHRS2 and what cellular functions does it perform?

DHRS2 is an NADPH-dependent oxidoreductase that belongs to the short-chain dehydrogenase/reductase (SDR) family. It primarily functions as a dicarbonyl reductase, catalyzing the reduction of various dicarbonyl compounds including 3,4-hexanedione, 2,3-heptanedione, and 1-phenyl-1,2-propanedione . At the cellular level, DHRS2 has several important functions:

• Acts as an enzymatic inactivator of reactive carbonyls involved in the covalent modification of cellular components

• Displays minor hydroxysteroid dehydrogenase activity toward bile acids such as ursodeoxycholic acid (UDCA)

• Attenuates MDM2-mediated p53/TP53 degradation, leading to p53 stabilization

• Reduces proliferation, migration, and invasion of cancer cells

• Decreases the production of reactive oxygen species (ROS) in cancer cells

DHRS2 is primarily localized in the mitochondrion matrix, though a minor fraction can be translocated to the nucleus after cleavage of the targeting signal .

What are the common applications for DHRS2 antibodies in research?

DHRS2 antibodies are versatile research tools employed in multiple experimental methodologies:

Researchers should select antibodies based on their specific application requirements and validate them in their experimental system .

How should DHRS2 antibodies be stored and handled to maintain optimal activity?

For maintaining optimal antibody activity, adhere to these storage and handling guidelines:

• Store at -20°C in aliquots to minimize freeze-thaw cycles

• Most commercial DHRS2 antibodies are supplied in buffer containing 50% glycerol and stabilizers

• Avoid repeated freeze-thaw cycles as they can degrade antibody quality

• When working with the antibody, keep it on ice or at 4°C

• For long-term storage (>12 months), some manufacturers recommend -80°C

• Before immunofluorescence applications, centrifuge antibody vials briefly to collect solution at the bottom

Many DHRS2 antibodies remain stable for 12 months when stored properly at -20°C . When shipping is required, antibodies should be transported with ice packs and immediately stored at the recommended temperature upon receipt .

What are the differences between various types of DHRS2 antibodies available to researchers?

DHRS2 antibodies vary significantly in their properties and applications:

When selecting an antibody, researchers should consider the specific epitope recognized, as this affects detection efficiency of specific DHRS2 isoforms or post-translationally modified forms .

How can I validate the specificity of my DHRS2 antibody in experimental systems?

Comprehensive validation of DHRS2 antibodies requires a multi-faceted approach:

• Positive and negative control samples:

  • Use cell lines with known DHRS2 expression (high expression: SKOV3; low expression: OVCAR3, HO-8910)

  • Include DHRS2 knockout/knockdown samples as negative controls using shRNA targeting DHRS2

• Molecular weight verification:

  • The calculated molecular weight of DHRS2 is approximately 30 kDa

  • Note that the observed band size may not always match calculations due to post-translational modifications

  • When using Western blotting, verify band size against recombinant DHRS2 protein

• Cross-validation with multiple antibodies:

  • Use antibodies recognizing different epitopes of DHRS2 (e.g., N-terminal vs. C-terminal)

  • Compare results from polyclonal and monoclonal antibodies

• Subcellular localization confirmation:

  • DHRS2 should primarily localize to mitochondria with some nuclear staining

  • Use co-staining with mitochondrial markers to confirm proper localization

• Immunoprecipitation followed by mass spectrometry:

  • For ultimate validation, perform IP with the DHRS2 antibody followed by MS identification

For immunofluorescence validation, researchers should observe DHRS2 protein specifically at mitochondria, as confirmed in HepG2 cells with GTX123431 antibody .

What experimental considerations are important when using DHRS2 antibodies in cancer research?

Cancer research involving DHRS2 antibodies requires specific methodological considerations:

• Cell line selection:

  • DHRS2 expression varies significantly across cancer cell lines

  • High expression: SKOV3 (ovarian cancer), HepG2 (hepatoblastoma)

  • Low expression: OVCAR3, HO-8910 (ovarian cancer), HK1 (nasopharyngeal carcinoma)

  • Consider creating stable cell lines with DHRS2 overexpression or knockdown

• Functional assays:

  • Cell proliferation: Use EdU incorporation assays to measure DNA synthesis

  • Colony formation assays: Assess long-term proliferative capacity

  • Cell invasion assays: Evaluate invasive capability through Matrigel

  • Xenograft models: For in vivo validation of DHRS2 effects

• Mechanistic studies:

  • Examine p53 pathway components (MDM2, p21) when modulating DHRS2 expression

  • Assess EMT markers (E-cadherin) in migration/invasion studies

  • Consider ERCC1 expression for chemosensitivity studies

• Clinical correlation:

  • When studying patient samples, correlate DHRS2 expression with clinical parameters

  • Consider chromosome 14q11.2 status, as this region shows high-frequency loss of heterozygosity in many tumors

When designing experiments, remember that DHRS2 has been implicated as both a tumor suppressor and oncogene depending on cancer type, underscoring the importance of context-specific validation .

How do I troubleshoot inconsistent results when using DHRS2 antibodies in Western blotting experiments?

When troubleshooting Western blotting with DHRS2 antibodies, consider these specific issues:

• Unexpected band sizes:

  • DHRS2's calculated MW is 30 kDa, but observed bands may differ

  • Post-translational modifications or protein cleavage can alter migration

  • Nuclear translocation of DHRS2 involves cleavage of its targeting signal

  • Solution: Include positive control lysates with known DHRS2 expression

• Multiple bands:

  • May indicate splice variants or post-translational modifications

  • Can result from cross-reactivity with other SDR family members

  • Solution: Use antibodies targeting different epitopes to confirm specificity

• Weak or no signal:

  • Optimization guidance table:

  Parameter	Recommended Adjustments for DHRS2 Detection
  Antibody dilution	Start with 1:500-1:2000 for most DHRS2 antibodies
  Blocking buffer	5% non-fat milk in TBST is generally effective
  Incubation time	Overnight at 4°C for primary antibody
  Cell lysis buffer	Include protease inhibitors to prevent degradation
  Sample loading	20-40 μg of total protein per lane
  Detection method	ECL or fluorescence-based detection systems

• Background issues:

  • Increase washing frequency and duration

  • Optimize blocking conditions (try 5% BSA instead of milk)

  • Decrease antibody concentration if using too much

• Cell-specific considerations:

  • Verify DHRS2 expression in your cell type before experiments

  • For verified samples, refer to MCF-7 for Western blotting protocols

When possible, include both positive controls (cells with known DHRS2 expression) and negative controls (DHRS2 knockdown cells) in troubleshooting experiments .

What role does DHRS2 play in chemoresistance, and how can antibodies help study this mechanism?

DHRS2 has emerged as a significant factor in chemoresistance, particularly in colorectal cancer:

• DHRS2 upregulation in chemoresistant cells:

  • Proteomics studies have identified DHRS2 as significantly upregulated in oxaliplatin-resistant colorectal cancer cells (HCT116/Oxa)

  • Western blot analysis with DHRS2 antibodies can confirm this upregulation

• DHRS2-mediated resistance mechanisms:

  • DHRS2 regulates ERCC1 expression through a p53-dependent pathway

  • DHRS2 binds to MDM2, attenuating MDM2-mediated p53 degradation

  • This leads to p53 stabilization and increased transcriptional activity

  • DHRS2 also mediates epithelial-mesenchymal transition (EMT) by suppressing E-cadherin expression

• Experimental approaches using antibodies:

  • Knockdown experiments: Transfect cells with DHRS2 siRNA and measure chemosensitivity

  • Protein interaction studies: Use co-immunoprecipitation with DHRS2 antibodies to detect interactions with MDM2

  • EMT marker analysis: Use Western blotting to detect changes in E-cadherin expression

  • Signaling pathway analysis: Examine p53 and ERCC1 expression levels

• Research findings on DHRS2 and chemoresistance:

  Cancer Type	Drug Resistance	DHRS2 Effect	Detection Method	Reference
  Colorectal cancer	Oxaliplatin	DHRS2 knockdown sensitized resistant cells	Western blotting
  Gastric cancer	5-FU	DHRS2 downregulation conferred insensitivity	Antibody detection
  Acute myelogenous leukemia	Multiple drugs	DHRS2 aberrant expression associated with resistance	Antibody-based assays

Researchers can use DHRS2 antibodies to identify potential therapeutic targets for simultaneously addressing cancer metastasis and chemoresistance .

How can DHRS2 antibodies be utilized in RNA-protein interaction studies?

DHRS2 antibodies have proven valuable in studying RNA-protein interactions through RNA immunoprecipitation (RIP) assays:

• RIP assay protocol for DHRS2:

  • Bind 4 μg of anti-DHRS2 antibody to magnetic beads

  • Incubate with cell lysates at 4°C overnight

  • Use control IgG antibody (1:20 dilution) as negative control

  • Digest protein with proteinase K

  • Extract RNA and analyze by qRT-PCR

• Applications in cancer biology:

  • Used to investigate DHRS2 interactions with specific mRNAs like CHKα

  • Helps understand post-transcriptional regulation mechanisms

  • Can reveal novel DHRS2 functions beyond enzymatic activity

• RNA stability assays in conjunction with DHRS2 antibodies:

  • Treat cells with actinomycin D (5 μg/ml)

  • Collect samples at different time points (0h, 4h, 8h, 12h)

  • Extract RNA and analyze by qPCR

  • Compare RNA stability in cells with different DHRS2 expression levels

• Technical considerations:

  • Antibody selection is crucial; use antibodies validated for immunoprecipitation

  • Include appropriate controls (IgG, input samples)

  • Consider crosslinking to stabilize transient RNA-protein interactions

  • Verify DHRS2 pull-down efficiency by Western blotting before RNA analysis

These approaches have been instrumental in uncovering DHRS2's role in regulating mRNA stability and gene expression, expanding our understanding beyond its canonical enzymatic functions .

How does DHRS2 function as a tumor suppressor, and how can antibodies help elucidate this mechanism?

DHRS2 demonstrates tumor suppressor activity in multiple cancer types through several mechanisms:

• Inhibition of cell proliferation:

  • Overexpression of DHRS2 significantly suppresses cancer cell growth

  • In nasopharyngeal carcinoma (NPC), stable HK1-DHRS2 cells show reduced EdU-positive cells compared to control cells

  • Colony formation assays confirm decreased long-term proliferative capacity

• Inhibition of invasion and metastasis:

  • DHRS2 reduces the number of cells invading through Matrigel

  • Knockdown of DHRS2 enhances cancer cell invasion

  • These effects have been observed in ovarian cancer and NPC cells

• Regulation of p53 pathway:

  • DHRS2 attenuates MDM2-mediated p53 degradation

  • This leads to p53 stabilization and increased transcriptional activity

  • Resulting in accumulation of MDM2 and CDKN1A/p21

• Research approaches using antibodies:

  • Expression analysis: Western blotting with DHRS2 antibodies to compare expression levels across cell lines

  • Xenograft models: IHC staining of tumor tissues to confirm DHRS2 expression in vivo

  • Mechanistic studies: Co-IP to detect DHRS2-MDM2 interactions

• In vivo evidence:

  • Mice injected with HK1-DHRS2 cells displayed delayed tumor occurrence

  • At 11 days post-injection, tumor detection rates were 2/5 for DHRS2-overexpressing tumors versus 5/5 for controls

  • DHRS2 overexpression resulted in reduced tumor size and mass without affecting mouse body weight

DHRS2 antibodies provide essential tools for investigating these tumor suppressor mechanisms across different cancer types, enabling both expression analysis and mechanistic studies .

What methodological considerations are important when using DHRS2 antibodies for immunofluorescence studies?

Immunofluorescence with DHRS2 antibodies requires specific technical considerations:

• Sample preparation protocol:

  • Fixation: Use 2% paraformaldehyde in culture medium at 37°C for 30 minutes

  • This protocol has been validated for HepG2 cells when staining with GTX123431 antibody

  • Permeabilization conditions may vary depending on the cellular compartment targeted

• Antibody dilution optimization:

  • Start with 1:500 dilution for GTX123431 antibody

  • Titrate to determine optimal signal-to-noise ratio for your specific cell type

  • Include appropriate negative controls (secondary antibody only, isotype control)

• Mitochondrial localization visualization:

  • DHRS2 antibodies should detect the protein primarily in mitochondria

  • Consider co-staining with mitochondrial markers for confirmation

  • A minor fraction may be detected in the nucleus due to translocation

• Signal detection and image acquisition:

  • Use appropriate filters for your secondary antibody fluorophore

  • Capture z-stacks to fully visualize three-dimensional distribution

  • Include nuclear counterstain (e.g., Hoechst 33342) at appropriate concentration

• Common pitfalls and solutions:

  Problem	Potential Cause	Solution
  No signal	Insufficient permeabilization	Optimize detergent concentration and incubation time
  High background	Excessive antibody concentration	Increase dilution factor of primary antibody
  Non-specific staining	Cross-reactivity	Use antibodies validated for IF applications
  Weak mitochondrial signal	Fixation affecting epitope	Try alternative fixation methods (methanol, acetone)

When performing IF with DHRS2 antibodies, it's essential to verify subcellular localization patterns consistent with expected mitochondrial and occasional nuclear distribution .

How can researchers design effective knockdown and overexpression experiments to study DHRS2 function using antibodies for validation?

Designing effective genetic manipulation experiments for DHRS2 requires careful planning and validation:

• DHRS2 knockdown strategies:

  • siRNA approach: Multiple studies have successfully used siRNA targeting DHRS2

  • shRNA approach: shDHRS2 constructs have been validated in several cancer cell lines

  • Specific example: In C666-1 cells, shDHRS2 3# showed the best knockdown efficiency

• DHRS2 overexpression strategies:

  • Vector selection: pEZ-Lv105-DHRS2 has been validated for DHRS2 overexpression

  • Cell line examples: Stable overexpression has been achieved in HK1, OVCAR3, and HO-8910 cells

• Validation using antibodies:

  • Western blotting: Primary validation method to confirm expression changes

  • qRT-PCR: Complement protein-level changes with mRNA quantification

  • Immunofluorescence: Visualize changes in DHRS2 localization and expression

• Functional assays following manipulation:

  • Cell growth: Measure using CCK-8 assay or direct cell counting

  • DNA synthesis: Quantify with EdU incorporation assay

  • Migration and invasion: Assess using Transwell assays

  • Colony formation: Evaluate long-term proliferative capacity

• Experimental design considerations:

  Experiment Type	Control Selection	Antibody Application	Expected Outcome
  siRNA knockdown	Non-targeting siRNA	WB confirmation of knockdown	Enhanced growth/invasion in cancer cells
  shRNA stable knockdown	Non-targeting shRNA	WB and IF for expression/localization	Increased tumorigenicity in vivo
  Overexpression	Empty vector	WB confirmation of expression	Reduced proliferation and invasion
  In vivo xenografts	Vector control cells	IHC of tumor sections	Delayed tumor formation, reduced size

For rigorous validation, researchers should confirm protein-level changes using antibodies targeting different epitopes of DHRS2 to ensure specificity of manipulation .

What is the relationship between DHRS2 and the p53 pathway, and how can antibodies be used to investigate this interaction?

DHRS2 has a complex relationship with the p53 pathway that can be investigated using antibody-based techniques:

• DHRS2-MDM2-p53 interaction mechanism:

  • DHRS2 binds to MDM2, attenuating MDM2-mediated p53 degradation

  • This leads to p53 stabilization and increased transcriptional activity

  • Results in accumulation of downstream targets like MDM2 and CDKN1A/p21

• Experimental approaches using antibodies:

  • Co-immunoprecipitation: Use DHRS2 antibodies to pull down protein complexes and detect MDM2

  • Western blotting: Monitor p53, MDM2, and p21 levels after DHRS2 manipulation

  • Chromatin immunoprecipitation: Assess p53 binding to target promoters

• DHRS2 in p53-dependent chemoresistance:

  • In colorectal cancer, DHRS2 regulates ERCC1 through a p53-dependent pathway

  • DHRS2 silencing sensitized HCT116/Oxa cells to oxaliplatin by downregulating ERCC1

  • Western blotting showed p53 was decreased in si-DHRS2 HCT116/Oxa cells

• Context-dependent regulation:

  • In some cancer types, DHRS2 expression is decreased despite its role in stabilizing p53

  • This paradox may be explained by chromosome 14q11.2 loss of heterozygosity

  • Alternative pathways may override p53 activation in specific cellular contexts

• Antibody panel for p53 pathway investigation:

  Target Protein	Purpose in DHRS2-p53 Studies	Recommended Application
  DHRS2	Confirm expression manipulation	WB, IF, IHC
  p53	Monitor stabilization levels	WB, IP, IF
  MDM2	Detect interaction with DHRS2	Co-IP, WB
  p21	Assess downstream p53 activity	WB, IF
  ERCC1	Examine DNA repair pathway regulation	WB

This relationship between DHRS2 and p53 explains how DHRS2 can function as both a tumor suppressor and a mediator of chemoresistance in different contexts .

How should researchers properly design controls for DHRS2 antibody experiments?

Proper control design is critical for reliable DHRS2 antibody experiments:

• Positive controls for DHRS2 detection:

  • Cell lines with confirmed DHRS2 expression:

    • SKOV3 (ovarian cancer): high DHRS2 expression

    • HepG2 (hepatoblastoma): DHRS2 is upregulated after sodium butyrate treatment

    • MCF-7: validated for Western blotting with E-AB-19202 antibody

  • Recombinant DHRS2 protein: For antibody validation and band size confirmation

• Negative controls for specificity:

  • Genetic knockdown: Use siRNA or shRNA targeting DHRS2

  • DHRS2-negative cell lines: Most ovarian cancer cell lines (except SKOV3) show low expression

  • Isotype control antibodies: For immunoprecipitation and immunofluorescence

  • Secondary antibody only: To detect non-specific binding

• Application-specific controls:

  Application	Essential Controls	Purpose
  Western Blotting	Loading control (β-actin, GAPDH)	Normalize protein loading
  	Molecular weight marker	Confirm band size (expected ~30 kDa)
  Immunofluorescence	Secondary antibody only	Detect non-specific binding
  	DHRS2 knockdown cells	Validate signal specificity
  	Mitochondrial co-staining	Confirm subcellular localization
  Immunoprecipitation	IgG control	Identify non-specific binding
  	Input sample	Confirm presence in starting material
  RIP Assay	IgG antibody (1:20)	Control for non-specific RNA binding

• Validation across multiple methods:

  • Confirm DHRS2 expression changes at both protein (Western blot) and mRNA (qRT-PCR) levels

  • Use multiple antibodies targeting different epitopes when possible

  • Validate localization using both biochemical fractionation and imaging techniques

Implementing these comprehensive controls ensures reliable interpretation of DHRS2 antibody experimental results and helps distinguish true signals from technical artifacts .

What are the optimal conditions for detecting DHRS2 in different cellular compartments?

DHRS2 localizes to multiple cellular compartments, requiring specific detection approaches:

• Mitochondrial DHRS2 detection:

  • Primary localization: DHRS2 is predominantly found in the mitochondrion matrix

  • Immunofluorescence protocol:

    • Fixation: 2% paraformaldehyde/culture medium at 37°C for 30 min

    • Antibody: GTX123431 at 1:500 dilution has been validated

    • Co-staining: Consider mitochondrial markers like MitoTracker or TOM20

    • Permeabilization: Requires optimization to access mitochondrial matrix

• Nuclear DHRS2 detection:

  • Secondary localization: A minor fraction translocates to the nucleus after cleavage of targeting signal

  • Detection considerations:

    • Nuclear extraction protocols may be needed for biochemical analysis

    • Use nuclear counterstains (e.g., DAPI, Hoechst) to confirm nuclear localization

    • Consider nuclear/cytoplasmic fractionation for Western blotting

• Cell type-specific considerations:

  Cell Type	DHRS2 Expression	Predominant Localization	Validated Antibody
  HepG2	High (after sodium butyrate)	Mitochondrial and nuclear	GTX123431
  SKOV3	High (constitutive)	Primarily mitochondrial	Various
  Normal nasopharyngeal epithelial cells (NP69, NP460)	Higher than NPC cells	Mitochondrial	Various
  HCT116/Oxa	Upregulated vs. parental cells	Not specified	Various

• Technical optimization for subcellular detection:

  • Fixation: Optimize to preserve both mitochondrial and nuclear structure

  • Antibody concentration: May require different dilutions for different compartments

  • Image acquisition: Use confocal microscopy for precise localization

  • Signal amplification: Consider tyramide signal amplification for low abundance detection

Understanding DHRS2's dual localization is crucial for experimental design and interpretation, particularly when studying its non-enzymatic functions in the nucleus versus its enzymatic roles in mitochondria .

How can researchers quantitatively analyze DHRS2 expression in cancer tissues using antibody-based methods?

Quantitative analysis of DHRS2 expression in cancer tissues requires standardized approaches:

• Immunohistochemistry (IHC) protocol optimization:

  • Antibody selection: E-AB-19202 has been validated for IHC at 1:50-1:200 dilution

  • Antigen retrieval: Critical for formalin-fixed paraffin-embedded tissues

  • Detection system: Consider amplification methods for low expression

  • Counterstaining: Hematoxylin for nuclear visualization

• Scoring systems for DHRS2 expression:

  • Semi-quantitative scoring:

    • Staining intensity: 0 (negative), 1 (weak), 2 (moderate), 3 (strong)

    • Percentage of positive cells: 0-100%

    • H-score calculation: Intensity × percentage (range: 0-300)

  • Digital image analysis:

    • Use software to quantify optical density or immunoreactive area

    • Enables more objective and reproducible quantification

• Comparative analysis frameworks:

  Tissue Analysis Approach	Methodology	Quantification Method	Advantages
  Tumor vs. normal tissue	Paired sample analysis	Direct comparison of H-scores	Controls for patient variability
  TMA analysis	Multiple patient samples	Statistical comparison across groups	High throughput, reduced variability
  Correlation with clinical outcomes	Survival analysis	Kaplan-Meier with optimal cutoffs	Links expression to prognosis
  Subcellular distribution analysis	High-resolution imaging	Ratio of nuclear/mitochondrial staining	Insight into functional state

• Validation and quality control:

  • Include positive controls: Tissues with known DHRS2 expression (e.g., liver cancer)

  • Negative controls: Omit primary antibody on serial sections

  • Biological validation: Correlate IHC with other methods (Western blot, qRT-PCR)

  • Blind scoring: Have multiple observers score samples independently

• Clinical correlation considerations:

  • DHRS2 expression varies by cancer type with both tumor-suppressive and oncogenic roles reported

  • Consider chromosome 14q11.2 status, as this region shows high-frequency loss of heterozygosity

  • Correlate with markers of p53 pathway activation (MDM2, p21)

  • For chemoresistance studies, correlate with ERCC1 expression

These approaches enable robust quantitative analysis of DHRS2 expression in cancer tissues, facilitating comparisons across patients and correlation with clinical outcomes .

What novel applications of DHRS2 antibodies are emerging in cancer research?

Several innovative applications of DHRS2 antibodies are advancing cancer research:

• Biomarker development for chemoresistance prediction:

  • DHRS2 upregulation in oxaliplatin-resistant colorectal cancer suggests potential as a predictive biomarker

  • Antibody-based detection in pre-treatment biopsies could guide therapy selection

  • Combined detection with ERCC1 may improve predictive value

• Therapeutic target validation:

  • DHRS2 antibodies are being used to validate it as a potential therapeutic target

  • Simultaneous addressing of cancer metastasis and chemoresistance

  • Monitoring DHRS2 expression changes in response to experimental therapies

• RNA-protein interaction studies:

  • RNA immunoprecipitation with DHRS2 antibodies reveals non-canonical functions

  • Identification of mRNA targets regulated by DHRS2

  • Investigation of post-transcriptional regulatory mechanisms

• Single-cell analysis applications:

  • Adaptation of DHRS2 antibodies for mass cytometry (CyTOF)

  • Flow cytometry applications for heterogeneity assessment

  • Correlation with other cancer markers at single-cell resolution

• Pathway mapping applications:

  Pathway	DHRS2 Antibody Application	Cancer Relevance
  p53-MDM2 axis	Co-IP to detect interactions	Tumor suppression mechanisms
  EMT pathway	Monitor E-cadherin changes after DHRS2 manipulation	Metastasis and invasion
  DNA repair	Correlate with ERCC1 expression	Chemoresistance mechanisms
  Cell cycle regulation	Assess cyclin D1 levels in response to DHRS2	Growth inhibition mechanisms

• Combination therapy response prediction:

  • Using DHRS2 antibodies to stratify patients for clinical trials

  • Monitoring DHRS2 expression changes during treatment

  • Correlating with treatment outcomes in patient-derived xenograft models

These emerging applications highlight the versatility of DHRS2 antibodies beyond traditional detection methods, positioning them as valuable tools in translational cancer research .