KRT33B Antibody

What is KRT33B and why is it significant in research?

KRT33B (keratin 33B, also known as hair keratin type I Ha3-II) belongs to the family of keratins, which form the intermediate filament cytoskeleton of epithelial cells. It is classified as a type I (acidic) keratin and has been identified as an androgen receptor (AR) target gene. KRT33B has gained significance in research due to its regulation by testosterone and its potential role in tissue development. Most notably, studies have demonstrated that KRT33B is expressed in rat penis tissues and is directly regulated by the androgen-AR signaling pathway, suggesting its involvement in androgen-mediated development processes . Understanding KRT33B's cellular functions provides insights into hormone-regulated tissue growth mechanisms and epithelial cell biology.

What are the key characteristics of commercially available KRT33B antibodies?

Commercial KRT33B antibodies are primarily available as polyclonal antibodies raised in rabbits, with validated reactivity against human KRT33B. Key characteristics include:

These antibodies are generally supplied in liquid form, purified via antigen affinity methods, and are unconjugated, requiring appropriate secondary antibodies for detection .

How is KRT33B protein expression distributed across different tissues?

Based on available research data, KRT33B shows a specific tissue distribution pattern. It has been detected in:

• Human brain tissue (via Western blot)

• MCF-7 cells (breast cancer cell line)

• Human skin cancer tissue and prostate cancer tissue (via IHC)

• Rat penis tissue, specifically in urethral epithelial cells and cavernosum interstitial cells (via immunofluorescence)

Importantly, subcellular localization studies indicate that KRT33B protein primarily localizes to the cytoplasm, consistent with its role as an intermediate filament protein. This cytoplasmic localization has been confirmed through immunofluorescence staining in both MCF-7 cells and rat penis tissues . This distribution pattern suggests potential roles beyond traditional structural functions in specific tissues.

What are the optimal conditions for using KRT33B antibodies in Western blot applications?

For Western blot applications with KRT33B antibodies, researchers should consider the following optimized protocol based on published methodologies:

• Sample preparation:

  • Use 30 μg of total protein from tissues or cultured cells

  • Separate proteins using 10% SDS-PAGE at 4°C and 120 V for approximately 90 minutes

• Transfer and blocking conditions:

  • Transfer proteins to nitrocellulose membranes

  • Block membranes using Tris buffer solution with 0.05% Tween 20 containing 5% nonfat milk for 1 hour at room temperature

• Antibody incubation:

  • Primary antibody dilution: 1:500-1:2000 (optimize based on specific antibody and sample)

  • Incubate with primary antibody overnight at 4°C

  • Wash thoroughly with TBST

  • Incubate with peroxidase-conjugated secondary antibody for 1 hour at room temperature

• Detection:

  • Visualize using ECL detection system followed by exposure to X-ray film

  • Expected molecular weight: approximately 46 kDa

Always include appropriate positive controls such as MCF-7 cells or human brain tissue lysates, which have been validated to express detectable levels of KRT33B .

How should researchers optimize immunohistochemistry protocols for KRT33B detection in different tissue types?

For optimal KRT33B detection in tissue sections via immunohistochemistry, researchers should consider tissue-specific adaptations:

• Antigen retrieval methods:

  • Primary recommendation: TE buffer pH 9.0

  • Alternative method: Citrate buffer pH 6.0 (particularly useful for certain tissue types)

• Antibody dilution range:

  • Recommended dilution: 1:400-1:1600 (requires optimization based on tissue type and fixation method)

• Tissue-specific considerations:

  • For skin cancer and prostate cancer tissues: The standard protocol has been validated

  • For other tissue types: Preliminary titration experiments are recommended

• Controls and validation:

  • Positive controls: Include human skin or prostate tissues

  • Negative controls: Omit primary antibody or use isotype control

  • Specificity validation: Consider using tissues from KRT33B knockout models when available

It is crucial to note that antigen retrieval conditions significantly impact KRT33B detection sensitivity in formalin-fixed, paraffin-embedded tissues. For novel tissue types not previously reported, researchers should conduct preliminary experiments comparing both antigen retrieval methods to determine optimal conditions .

What considerations are important when designing immunofluorescence experiments with KRT33B antibodies?

When designing immunofluorescence experiments to detect KRT33B, researchers should consider:

• Cell/tissue preparation:

  • For cultured cells (e.g., MCF-7): Standard fixation with 4% paraformaldehyde followed by permeabilization with 0.1% Triton X-100

  • For tissue sections: Cryosections are preferable, but FFPE sections with appropriate antigen retrieval can also be used

• Antibody parameters:

  • Recommended dilution range: 1:50-1:500 (requires optimization)

  • Incubation conditions: Typically overnight at 4°C for primary antibody

• Co-localization studies:

  • KRT33B primarily shows cytoplasmic localization

  • For androgen signaling studies, co-staining with anti-AR antibodies can be informative, as these proteins show distinct subcellular localization patterns (KRT33B in cytoplasm, AR primarily in nucleus)

• Controls and visualization:

  • Include appropriate positive controls (MCF-7 cells)

  • Consider counterstaining with DAPI to visualize nuclei

  • For tissue architecture delineation in complex samples, additional markers may be useful (e.g., smooth muscle actin, von Willebrand factor as used in rat penis studies)

Double-labeling immunofluorescence has proven particularly valuable for examining the relationship between KRT33B and regulatory proteins like androgen receptor, as demonstrated in studies of rat penis tissue .

How is KRT33B expression regulated at the molecular level?

Research has revealed that KRT33B is directly regulated by the androgen-AR signaling pathway through the following mechanisms:

• Transcriptional regulation:

  • Androgen receptor (AR) directly binds to androgen response elements (AREs) in the KRT33B promoter

  • ChIP assays have identified a putative ARE in the approximately 2.4-kb sequence region upstream of the TATA box of the KRT33B gene

• Hormone responsiveness:

  • Testosterone treatment induces KRT33B expression in a dose-dependent manner in rat penis tissue

  • Anti-androgen treatment (flutamide) dramatically reduces KRT33B expression, confirming androgen-dependency

• Molecular verification:

  • siRNA knockdown of AR significantly reduces KRT33B expression in multiple cell lines (HepG2 and MHCC-97L), demonstrating the direct regulatory relationship

  • ChIP assays have confirmed direct AR binding to KRT33B promoter AREs

This regulatory mechanism appears to be tissue-specific and developmentally regulated, with particularly strong effects observed during puberty-associated tissue development. The identification of KRT33B as an androgen-responsive gene provides valuable insights into tissue-specific hormone action mechanisms .

What is the relationship between KRT33B and androgen receptor signaling in development and disease models?

The relationship between KRT33B and androgen receptor (AR) signaling represents a complex interplay with implications for both development and disease:

• Developmental context:

  • In rat models, KRT33B has been identified as a novel AR target gene involved in testosterone-stimulated penis growth during puberty

  • Microarray analysis revealed KRT33B as one of the most responsive genes to testosterone treatment during development

• Expression correlation:

  • KRT33B and AR are co-expressed in specific tissues (e.g., penis urethra epithelial cells and cavernosum interstitial cells)

  • KRT33B expression patterns follow testosterone-induced AR activation

• Signaling dynamics:

  • Testosterone treatment upregulates both AR protein expression and its target gene KRT33B

  • This creates a positive feedback loop that potentially amplifies androgen signaling effects during development

• Potential disease implications:

  • While direct evidence in pathological contexts is limited, the detection of KRT33B in cancer tissues (skin and prostate cancer) suggests potential involvement in disease processes

  • The androgen-dependent regulation suggests KRT33B might play roles in hormone-responsive cancers

These findings suggest KRT33B may serve as a molecular marker for androgen action in both physiological development and potentially in pathological conditions. Its tissue-specific expression pattern indicates specialized functions beyond the structural roles typically associated with keratin proteins .

What are the current methodological approaches for studying KRT33B protein interactions and modifications?

Advanced research into KRT33B protein interactions and modifications employs several sophisticated methodological approaches:

• Protein-protein interaction studies:

  • Co-immunoprecipitation (Co-IP) using KRT33B antibodies to identify interacting partners

  • Proximity ligation assays (PLA) to visualize protein interactions in situ

  • Yeast two-hybrid screening to identify novel binding partners

• Post-translational modification analysis:

  • Phosphorylation site mapping using phospho-specific antibodies or mass spectrometry

  • Ubiquitination analysis to assess protein stability and turnover

  • Site-directed mutagenesis of potential modification sites to assess functional consequences

• Structural studies:

  • Recombinant protein expression and purification for in vitro structural analysis

  • Protein domain mapping to determine functional regions involved in specific interactions

• Functional genomics approaches:

  • CRISPR-Cas9 genome editing to generate KRT33B knockout or mutant models

  • siRNA or shRNA knockdown to assess loss-of-function effects

  • ChIP-seq analysis to comprehensively map transcription factor binding sites in the KRT33B promoter region, as demonstrated in the identification of AR binding sites

These methodologies provide complementary approaches to understand KRT33B's role in cellular processes and signaling pathways. When designing such studies, researchers should consider the cell type-specific expression patterns of KRT33B and select appropriate experimental systems accordingly .

What are common challenges in detecting KRT33B and how can researchers overcome them?

Researchers commonly encounter several challenges when working with KRT33B antibodies:

• Low signal intensity:

  • Challenge: KRT33B expression levels vary significantly between tissues and may be low in certain samples

  • Solution: Optimize antibody concentration (testing dilutions from 1:50-1:500 for IF/ICC and 1:400-1:1600 for IHC); use enhanced detection methods like signal amplification systems; increase protein loading for Western blots (up to 30-50 μg)

• Non-specific binding:

  • Challenge: Some antibodies may show cross-reactivity with other keratin family members due to sequence homology

  • Solution: Validate antibody specificity using positive controls (MCF-7 cells, human brain tissue); include appropriate negative controls; consider using siRNA knockdown to confirm specificity

• Inconsistent results between applications:

  • Challenge: An antibody performing well in WB may not work optimally in IHC or IF

  • Solution: Each application requires independent optimization; adjust fixation methods, antigen retrieval conditions (compare TE buffer pH 9.0 vs. citrate buffer pH 6.0), and blocking conditions

• Tissue-specific detection issues:

  • Challenge: KRT33B detection varies across tissue types

  • Solution: Refer to validated positive tissue types (human skin cancer tissue, human prostate cancer tissue, MCF-7 cells) for protocol development; optimize antigen retrieval specifically for each tissue type

Importantly, each new experimental system requires titration of the antibody to determine optimal working conditions. Sample-dependent variability means researchers should not assume identical protocols will work across different tissue types or experimental conditions .

How can researchers verify KRT33B antibody specificity in their experimental systems?

Verifying antibody specificity is crucial for reliable KRT33B detection. Researchers should employ multiple validation approaches:

• Molecular weight verification:

  • Confirm detection at the expected molecular weight (approximately 46 kDa) in Western blot applications

  • Be aware of potential post-translational modifications that may cause molecular weight shifts

• Positive and negative controls:

  • Positive controls: Include validated KRT33B-expressing samples (MCF-7 cells, human brain tissue)

  • Negative controls: Include samples from tissues known not to express KRT33B or use genetic knockdown models

• Genetic manipulation approaches:

  • siRNA/shRNA knockdown of KRT33B to demonstrate decreased antibody signal

  • Overexpression of tagged KRT33B to confirm antibody detection

  • CRISPR-Cas9 knockout systems for complete elimination of target protein

• Peptide competition assays:

  • Pre-incubate antibody with immunizing peptide prior to application

  • Specific binding should be blocked by the peptide, resulting in signal loss

• Orthogonal detection methods:

  • Compare protein detection with mRNA expression data

  • Use multiple antibodies targeting different epitopes of KRT33B

  • Employ mass spectrometry to confirm protein identity in immunoprecipitated samples

Thorough validation is particularly important when studying KRT33B in novel experimental systems or tissue types not previously characterized for KRT33B expression .

What considerations are important when analyzing KRT33B expression in hormone-responsive tissues?

When analyzing KRT33B expression in hormone-responsive tissues, researchers should consider several important factors:

• Hormonal status control:

  • Document and control the hormonal status of experimental animals or cell cultures

  • For animal studies, consider castration and hormone replacement paradigms as demonstrated in rat penis growth studies

  • For cell cultures, use hormone-depleted serum conditions before hormone treatment

• Time-course considerations:

  • KRT33B expression changes dynamically in response to hormonal stimulation

  • Design experiments with appropriate time points to capture both early and late responses

  • In developmental studies, carefully document age and developmental stage

• Dose-response relationships:

  • Testosterone regulation of KRT33B shows dose-dependent characteristics

  • Include multiple hormone concentrations in experimental design to establish dose-response curves

• Pathway validation:

  • Include AR antagonists (e.g., flutamide) as controls to confirm AR-mediated effects

  • Consider the potential impact of other hormone signaling pathways that might cross-talk with androgen signaling

  • Measure AR expression levels alongside KRT33B to assess correlation

• Tissue heterogeneity:

  • Consider cell type-specific expression patterns within complex tissues

  • Use double-labeling approaches with cell type-specific markers to identify exactly which cells express KRT33B

These considerations help ensure that observed changes in KRT33B expression are correctly attributed to specific hormonal mechanisms, particularly important in complex developmental processes or disease models where multiple signaling pathways may be altered .

What are the potential applications of KRT33B as a biomarker in clinical and experimental settings?

Based on current research findings, KRT33B shows promise as a biomarker in several contexts:

• Androgen-responsive tissue development:

  • KRT33B has been identified as a direct androgen receptor target gene

  • Its expression correlates with testosterone-stimulated tissue growth, particularly in developmental contexts

  • Potential application in monitoring normal and abnormal androgen-dependent development

• Cancer research applications:

  • Detection in multiple cancer tissues including skin cancer and prostate cancer suggests potential relevance

  • Given its androgen responsiveness, KRT33B may have specific utility in hormone-dependent cancers

  • Could serve as a marker for assessing AR pathway activity in cancer tissues

• Experimental systems:

  • As a downstream target of androgen signaling, KRT33B expression could serve as a readout for AR pathway activation in experimental models

  • Useful for evaluating the efficacy of androgen/anti-androgen treatments in research settings

• Developmental biology:

  • Monitoring KRT33B expression during critical developmental windows may provide insights into hormone-dependent tissue differentiation processes

  • Potentially useful in studying disorders of sexual development or other androgen-related developmental conditions

Future research should systematically evaluate KRT33B expression across a broader range of normal and pathological tissues to fully establish its utility as a biomarker in specific clinical or research applications .

How does KRT33B research interface with broader studies of intermediate filament biology?

KRT33B research contributes to the broader understanding of intermediate filament biology in several significant ways:

• Specialized keratin function beyond structural roles:

  • Traditional views of keratins focus on their structural roles in maintaining cellular integrity

  • KRT33B research reveals hormone-responsive regulation and tissue-specific expression patterns suggesting specialized functions beyond structural support

  • This expands our understanding of the diverse roles keratins play in cellular processes

• Transcriptional regulation of tissue-specific keratins:

  • The identification of direct androgen receptor regulation of KRT33B provides insights into how tissue-specific keratin expression is controlled

  • This regulatory mechanism may serve as a model for understanding how other specialized keratins are regulated in different tissues

• Intermediate filaments in cell signaling:

  • KRT33B's response to hormonal signals suggests intermediate filaments may function as downstream effectors in signaling pathways

  • This challenges the view of intermediate filaments as merely passive structural components

  • Raises questions about how changes in cytoskeletal composition might influence cell behavior and tissue development

• Evolutionary specialization of keratin proteins:

  • KRT33B belongs to the hair keratin subfamily, but its expression in non-hair tissues suggests evolutionary repurposing

  • This provides insights into how specialized keratin functions may have evolved from ancestral structural roles

These interfaces highlight how focused studies on specific keratin proteins like KRT33B contribute to redefining our understanding of intermediate filament biology, moving beyond structural roles to encompass regulatory functions in diverse cellular processes .