dhrs7cb Antibody

What is DHRS7B and why is it significant for research?

DHRS7B (Dehydrogenase/reductase SDR family member 7B) is a member of the short-chain dehydrogenase/reductase (SDR) family involved in the metabolism of steroids and retinoids. It shares functional similarities with DHRS7 (also known as NET50, retSDR4, and SDR34C1), which localizes predominantly in the nuclear envelope . DHRS7B is significant for research because SDR family proteins play critical roles in various cellular processes, including steroid conversion and potentially nuclear size regulation, which has implications in cancer progression . The protein's expression patterns across tissues and its potential role in disease states make antibodies against it valuable research tools for investigating cellular localization, protein interactions, and functional studies.

What are the common applications for DHRS7B antibodies in research?

DHRS7B antibodies are primarily used in the following research applications:

• Immunohistochemistry (IHC) - For detection of DHRS7B in tissue samples

• Western Blotting (WB) - For protein expression analysis

• Immunocytochemistry and Immunofluorescence (ICC-IF) - For subcellular localization studies

• ELISA - For quantitative detection in solution

These antibodies enable researchers to study protein expression patterns, subcellular localization (particularly in relation to the nuclear envelope), and potential changes in expression during disease progression or in response to experimental treatments .

How do I select the appropriate DHRS7B antibody for my research application?

When selecting a DHRS7B antibody, consider these key factors:

• Validated applications: Ensure the antibody has been validated for your specific application (IHC, WB, ICC-IF). Look for antibodies that have undergone rigorous validation procedures .

• Species reactivity: Confirm the antibody recognizes DHRS7B in your species of interest. Available antibodies may target human DHRS7B but have varying cross-reactivity with other species .

• Clonality:

  • Polyclonal antibodies (like those available from Atlas Antibodies and Biomatik) offer high sensitivity and recognize multiple epitopes

  • Monoclonal antibodies provide higher specificity for a single epitope and greater batch-to-batch consistency

• Specificity verification: Review the validation data showing minimal cross-reactivity with other proteins. Some manufacturers use protein microarray technology to ensure monospecificity, which is crucial for obtaining reliable results .

• Format and conjugation: Select appropriate formats (purified, conjugated) based on your experimental needs .

What are the optimal protocols for using DHRS7B antibodies in Western blotting?

For optimal Western blotting results with DHRS7B antibodies:

• Sample preparation:

  • Use appropriate lysis buffers containing protease inhibitors

  • Consider subcellular fractionation to enrich nuclear envelope proteins if studying localization

• Protein loading and separation:

  • Load 20-50 μg of total protein per lane

  • Use 10-12% SDS-PAGE gels for optimal separation

• Transfer and blocking:

  • Transfer to PVDF or nitrocellulose membranes

  • Block with 5% non-fat milk or BSA in TBST for 1 hour at room temperature

• Antibody incubation:

  • Primary antibody dilution: Typically 1:500-1:1000 (optimize based on manufacturer's recommendation)

  • Incubate overnight at 4°C

  • Secondary antibody: Use species-appropriate HRP-conjugated antibody at 1:5000-1:10000 dilution

• Detection and controls:

  • Include positive controls such as prostate tissue lysates or LNCaP cell line extracts (which express DHRS7/DHRS7B)

  • Include loading controls (β-actin, GAPDH)

  • Use enhanced chemiluminescence (ECL) for detection

• Troubleshooting:

  • For weak signals, increase antibody concentration or extend incubation time

  • For high background, increase blocking time or washing steps

Validation techniques should confirm antibody specificity through peptide competition assays or knockout/knockdown controls .

How can I optimize immunohistochemistry protocols for DHRS7B detection in tissue samples?

For optimal IHC protocols with DHRS7B antibodies:

• Tissue fixation and processing:

  • Use 10% neutral buffered formalin for fixation (12-24 hours)

  • Paraffin embedding followed by 4-6 μm sectioning

• Antigen retrieval:

  • Heat-induced epitope retrieval (HIER) in citrate buffer (pH 6.0) or EDTA buffer (pH 9.0)

  • Pressure cooker or microwave methods (20 minutes)

• Blocking and antibody incubation:

  • Block endogenous peroxidase with 3% H₂O₂

  • Block non-specific binding with 5-10% normal serum

  • Primary antibody dilution: Start with 1:100-1:200 (optimize as needed)

  • Incubate in a humidified chamber overnight at 4°C

• Detection system:

  • Use polymer detection systems for enhanced sensitivity

  • DAB (3,3'-diaminobenzidine) as chromogen

  • Counterstain with hematoxylin

• Controls and validation:

  • Include positive control tissues known to express DHRS7B

  • Include negative controls (omitting primary antibody)

  • Consider using tissues with differential expression (e.g., high-grade vs. low-grade prostate cancer)

• Signal interpretation:

  • Look for specific subcellular localization patterns, particularly nuclear envelope staining

  • Score staining intensity and percentage of positive cells

How do I validate the specificity of a DHRS7B antibody for my research?

Validating DHRS7B antibody specificity is crucial for reliable research findings. Employ these methods:

• Peptide competition assay:

  • Pre-incubate the antibody with excess immunizing peptide

  • Compare results with and without peptide blocking

  • Specific signals should disappear in the blocked sample

• Genetic validation:

  • Use DHRS7B knockdown (siRNA/shRNA) or knockout (CRISPR/Cas9) cells/tissues

  • Compare antibody signal between wild-type and knockdown/knockout samples

  • Specific antibodies will show reduced/absent signal in knockdown/knockout samples

• Orthogonal validation:

  • Correlate protein detection with mRNA expression

  • Use multiple antibodies targeting different epitopes of DHRS7B

  • Compare results across different detection methods (WB, IHC, IF)

• Protein microarray screening:

  • Test against human proteome microarrays like HuProt™ to detect cross-reactivity

  • Select antibodies shown to be monospecific through such screening

• Independent expression system validation:

  • Test in recombinant expression systems (e.g., transfected cells)

  • Compare signals between transfected and non-transfected cells

These validation approaches help ensure that your findings are based on specific detection of DHRS7B rather than cross-reactivity with other proteins .

How can DHRS7B antibodies be used to study nuclear size regulation in cancer research?

Research has shown that DHRS7 (related to DHRS7B) is involved in nuclear size regulation, particularly in prostate cancer models . To study this connection:

• Nuclear morphology analysis:

  • Use immunofluorescence with DHRS7B antibodies combined with nuclear stains (DAPI/Hoechst)

  • Employ high-content imaging to quantify nuclear size parameters

  • Compare DHRS7B expression levels with nuclear size measurements

• Experimental manipulation:

  • Modulate DHRS7B expression through knockdown/overexpression

  • Monitor nuclear size changes using standardized nuclear morphometry

  • Based on DHRS7 research, knockdown may increase nuclear size while overexpression may decrease it

• Co-localization studies:

  • Combine DHRS7B antibodies with markers of the nuclear envelope

  • Assess potential co-localization with lamins, nuclear pore complexes, or other NET proteins

  • Evaluate structural integrity of the nuclear envelope

• Cancer progression models:

  • Compare DHRS7B expression and nuclear size across cancer progression models

  • Use tissue microarrays with varying grades of cancer

  • Correlate DHRS7B levels with clinical parameters and nuclear size changes

• Drug response studies:

  • Test compounds that may restore normal nuclear size (similar to estradiol propionate in DHRS7 studies)

  • Monitor effects on DHRS7B localization and expression

  • Assess cancer cell migration and invasion in relation to nuclear size changes

This research approach may help elucidate whether DHRS7B, like DHRS7, plays a role in nuclear size regulation and whether targeting this pathway could have therapeutic implications.

What are the considerations for using DHRS7B antibodies in combination with other antibodies for co-localization studies?

When designing co-localization experiments with DHRS7B antibodies:

• Antibody compatibility:

  • Select primary antibodies raised in different host species (e.g., rabbit anti-DHRS7B with mouse anti-lamin)

  • Ensure secondary antibodies have minimal cross-reactivity with non-target species

• Spectral considerations:

  • Choose fluorophores with minimal spectral overlap

  • Include single-stained controls for spectral unmixing if needed

  • Consider sequential imaging rather than simultaneous acquisition if bleed-through is a concern

• Fixation optimization:

  • Different antibodies may require different fixation methods

  • Test multiple fixation protocols (PFA, methanol, acetone)

  • Optimize fixation time and temperature for preserving both antigens

• Signal amplification:

  • For weak signals, consider tyramide signal amplification (TSA)

  • Balance signal intensities between markers to facilitate co-localization analysis

• Controls and quantification:

  • Include negative controls for each antibody

  • Use positive controls with known co-localization patterns

  • Employ quantitative co-localization analysis (Pearson's coefficient, Manders' overlap)

• Subcellular context:

  • For nuclear envelope studies, combine with lamin antibodies

  • For ER/Golgi studies, combine with organelle markers

  • For steroid metabolism studies, combine with other SDR family members

How does DHRS7B expression correlate with disease progression and what are the methodological considerations for such studies?

To investigate DHRS7B expression in disease progression:

• Tissue selection and processing:

  • Use matched normal and diseased tissues

  • Consider tissue microarrays for high-throughput analysis

  • Ensure consistent processing across all samples

• Quantitative expression analysis:

  • Implement standardized scoring systems for IHC (H-score, Allred score)

  • Use digital pathology and automated image analysis for objective quantification

  • Correlate protein expression with mRNA levels (by qRT-PCR or RNA-seq)

• Clinical correlation methodology:

  • Develop clear inclusion/exclusion criteria for patient samples

  • Collect comprehensive clinical data (diagnosis, stage, grade, treatment, outcomes)

  • Use appropriate statistical methods for correlation analysis

• Comparative expression studies:

  • Based on DHRS7 research in prostate cancer, investigate whether DHRS7B shows similar expression patterns

  • DHRS7 is highly expressed in normal prostate but decreases in higher-grade cancers

  • Determine if DHRS7B follows similar trends or has distinct expression patterns

• Methodological standards:

  • Use multiple antibody clones/lots to confirm findings

  • Include appropriate positive and negative controls

  • Validate findings with orthogonal techniques (Western blot, qRT-PCR)

• Prognostic value assessment:

  • Correlate expression levels with patient survival data

  • Use multivariate analysis to control for confounding factors

  • Consider time-dependent changes in expression

This approach allows for rigorous evaluation of DHRS7B as a potential biomarker, similar to how DHRS7 has been investigated in prostate cancer progression.

What are common challenges when working with DHRS7B antibodies and how can they be addressed?

How should researchers interpret conflicting data when using different DHRS7B antibodies?

When faced with conflicting results from different DHRS7B antibodies:

• Epitope differences:

  • Map the epitopes recognized by each antibody

  • Different epitopes may be differentially accessible in various experimental conditions

  • Post-translational modifications may affect epitope recognition

• Antibody validation status:

  • Prioritize results from antibodies with more extensive validation

  • Consider antibodies validated through protein microarrays or knockout models

  • Review published literature using the same antibodies

• Experimental conditions:

  • Standardize all experimental conditions between antibodies

  • Test all antibodies simultaneously under identical conditions

  • Vary conditions systematically to identify factors affecting antibody performance

• Orthogonal methods:

  • Confirm findings with non-antibody-based methods (mRNA analysis, mass spectrometry)

  • Use genetic approaches (overexpression, knockdown) to validate findings

  • Consider functional assays relevant to DHRS7B activity

• Resolution strategies:

  • Develop a consensus approach using multiple antibodies

  • Weight evidence based on validation quality

  • Report discrepancies transparently in publications

  • Consult with antibody manufacturers about potential issues

How can researchers distinguish between DHRS7 and DHRS7B in their experiments?

Distinguishing between these related proteins requires careful experimental design:

• Antibody specificity:

  • Select antibodies raised against unique regions with minimal sequence homology

  • Validate specificity using recombinant proteins of both DHRS7 and DHRS7B

  • Perform peptide competition assays with specific peptides for each protein

• Expression pattern analysis:

  • Compare with known tissue-specific expression patterns (DHRS7 is highly expressed in prostate)

  • Use tissues/cell lines with differential expression of the two proteins

  • Correlate with mRNA expression data for both genes

• Molecular weight discrimination:

  • Optimize gel conditions to separate proteins based on molecular weight differences

  • Use high-resolution gels (gradient gels) for better separation

  • Include recombinant protein standards for both proteins

• Genetic approaches:

  • Use specific siRNA/shRNA for each protein and confirm antibody specificity

  • Create specific knockout models for each gene

  • Perform rescue experiments with specific constructs

• Functional discrimination:

  • Design assays based on known functional differences

  • If studying nuclear size regulation, note that DHRS7 has been shown to affect this parameter

  • Consider substrate specificity differences if performing enzymatic assays

How can DHRS7B antibodies be incorporated into high-throughput screening methodologies?

For high-throughput applications involving DHRS7B antibodies:

• Automated immunoassay platforms:

  • Adapt ELISA protocols for robotics-based liquid handling systems

  • Develop multiplexed assays targeting DHRS7B alongside other relevant proteins

  • Utilize high-density plate formats (384/1536-well) for screening efficiency

• High-content imaging:

  • Combine DHRS7B immunofluorescence with automated microscopy

  • Develop quantitative image analysis pipelines for nuclear morphology

  • Implement machine learning for pattern recognition in subcellular localization

• Reverse phase protein arrays (RPPA):

  • Use validated DHRS7B antibodies in RPPA format for multiplexed patient sample analysis

  • Correlate with clinical outcomes across large sample cohorts

  • Integrate with other proteomic data

• Compound screening approaches:

  • Screen for modulators of DHRS7B expression or localization

  • Develop reporter assays based on DHRS7B activity or expression

  • Use similar approaches to those used for DHRS7, which identified estradiol propionate as a compound affecting nuclear size

• Quality control considerations:

  • Implement rigorous controls for antibody lot consistency

  • Include calibration standards across plates/batches

  • Utilize reference cell lines with known DHRS7B expression levels

What are the considerations for using DHRS7B antibodies in studying protein-protein interactions?

When investigating DHRS7B protein interactions:

• Co-immunoprecipitation (Co-IP) optimization:

  • Test multiple lysis buffers to preserve interactions

  • Consider crosslinking to stabilize transient interactions

  • Include appropriate controls (IgG, reverse Co-IP)

  • Validate antibody efficiency for immunoprecipitation

• Proximity ligation assay (PLA):

  • Combine DHRS7B antibody with antibodies against potential interacting partners

  • Optimize probe dilutions and reaction conditions

  • Include negative controls (non-interacting proteins)

  • Quantify PLA signals using appropriate image analysis

• FRET/BRET approaches:

  • Design constructs that retain native interaction capabilities

  • Consider the impact of tags on protein localization and function

  • Use antibodies for validation of expression and localization

• Mass spectrometry validation:

  • Use antibodies for pull-down experiments followed by MS

  • Compare with direct AP-MS approaches

  • Validate key interactions with orthogonal methods

• Nuclear envelope interactome:

  • Focus on potential interactions with nuclear envelope proteins

  • Investigate whether DHRS7B, like DHRS7, interacts with components involved in nuclear size regulation

  • Consider the implications for nuclear envelope integrity and function

How might DHRS7B antibodies contribute to understanding the potential therapeutic implications of targeting this protein in disease?

Using DHRS7B antibodies to explore therapeutic potential:

• Biomarker development:

  • Evaluate DHRS7B expression across disease progression

  • Determine if DHRS7B levels correlate with treatment response

  • Develop standardized IHC protocols for potential clinical use

  • Based on DHRS7 findings, investigate whether DHRS7B could serve as a prostate cancer biomarker

• Target validation:

  • Use antibodies to confirm target engagement of experimental compounds

  • Monitor changes in DHRS7B expression, localization, or modifications following treatment

  • Correlate with functional outcomes (e.g., nuclear size, cell migration)

• Companion diagnostics potential:

  • Investigate whether DHRS7B expression predicts response to specific therapies

  • Similar to how DHRS7 levels might predict response to estrogen-based treatments in prostate cancer

  • Develop quantitative assays suitable for clinical implementation

• Mechanism-of-action studies:

  • Use antibodies to track DHRS7B during drug treatment

  • Identify downstream effectors through proteomics approaches

  • Investigate pathway alterations in response to DHRS7B modulation

• Therapeutic antibody development considerations:

  • Evaluate the potential of antibodies themselves as therapeutics

  • Assess internalization potential if considering antibody-drug conjugates

  • Investigate whether DHRS7B is accessible in disease contexts

These approaches can help determine whether DHRS7B, like its related protein DHRS7, might represent a viable therapeutic target or biomarker in specific disease contexts .