KRT8 Antibody

What is KRT8 and why is it important in epithelial tissue research?

KRT8 (keratin 8) belongs to the type II (basic) subfamily of high molecular weight cytokeratins and typically exists in combination with cytokeratin 18 (KRT18). It functions as a crucial intermediate filament component in simple epithelia, including glandular epithelium of the thyroid, female breast, gastrointestinal tract, respiratory tract, and urogenital tract including transitional epithelium .

KRT8 is essential for maintaining cellular structural integrity while also participating in signal transduction and cellular differentiation processes . Together with KRT19, it helps link the contractile apparatus to dystrophin at the costameres of striated muscle . The importance of KRT8 in epithelial research stems from its tissue-specific expression pattern, making it valuable for studying epithelial differentiation, transformation, and carcinogenesis.

What are the most common applications for KRT8 antibodies in research?

KRT8 antibodies are versatile tools employed across multiple experimental techniques:

These applications are particularly valuable in cancer research, where KRT8 serves as a biomarker for adenocarcinomas and certain squamous carcinomas . Additionally, KRT8 antibodies are useful for studying epithelial-to-mesenchymal transition (EMT) in cancer progression and metastasis .

How should I select between monoclonal and polyclonal KRT8 antibodies?

The selection depends on your specific experimental needs:

Monoclonal antibodies (e.g., KRT8/803, UMAB1, KRT8/2174R):

• Offer high specificity targeting a single epitope

• Provide consistent lot-to-lot reproducibility

• Ideal for detecting specific KRT8 forms or modifications

• Excellent for immunohistochemistry applications with less background

Polyclonal antibodies:

• Recognize multiple epitopes on KRT8 protein

• Potentially higher sensitivity for low abundance targets

• Better for protein detection after denaturation (e.g., Western blot)

• May show higher cross-reactivity with related keratins

When studying KRT8 alongside KRT18, consider cocktail antibodies like KRT8/803 + KRT18/835 that recognize both proteins simultaneously, which is useful for detecting simple epithelia and adenocarcinomas .

What species reactivity should I consider when selecting a KRT8 antibody?

Species reactivity is crucial for experimental validity. Based on the search results, many commercially available KRT8 antibodies demonstrate cross-reactivity with multiple species:

When conducting comparative or translational studies across multiple species, select antibodies with verified cross-reactivity. For species not explicitly listed, sequence homology analysis and validation experiments should be performed before proceeding with full-scale studies .

How does phosphorylation of KRT8 affect its function and antibody detection?

KRT8 phosphorylation represents a critical post-translational modification that significantly alters its functional properties. Research shows that phosphorylation of KRT8 on Ser43 by overloading-activated RHOA-PKN (protein kinase N) impedes trafficking of Golgi resident small GTPase RAB33B, suppressing autophagosome initiation and contributing to intervertebral disc degeneration (IDD) .

For antibody detection considerations:

• Phospho-specific antibodies are required to detect specific phosphorylated forms of KRT8

• Standard KRT8 antibodies may show altered binding affinity to heavily phosphorylated KRT8

• Dephosphorylation treatments prior to immunodetection may be necessary in certain contexts

When studying KRT8 phosphorylation, it's advisable to use both phospho-specific and total KRT8 antibodies to compare relative proportions of modified versus unmodified protein .

What role does KRT8 play in mitochondrial homeostasis and how can this be studied?

KRT8 has been shown to attenuate necrotic cell death by facilitating mitophagy under oxidative stress conditions. Research demonstrates that KRT8 decreases the generation of paraquat-induced mitochondrial reactive oxygen species (ROS) in retinal pigment epithelial (RPE) cells .

Methodological approach for studying KRT8's role in mitochondrial homeostasis:

• Visualization techniques:

  • Co-localization studies using KRT8 antibodies with mitochondrial markers (MitoTracker)

  • Live-cell imaging with GFPSpark-tagged KRT8 and mitochondrial trackers

• Functional assays:

  • Mitochondrial ROS measurement using MitoSOX

  • Mitochondrial fission analysis via time-lapse microscopy

  • Calcium ion accumulation in mitochondria under stress conditions

• Genetic manipulation:

  • KRT8 knockdown and overexpression studies

  • Analysis of mitophagy flux using LC3-II markers

  • Assessment of mitochondrial dynamics proteins (DRP1, MFN1/2)

The research indicates that KRT8 is present at sites of mitochondrial fission in RPE cells under oxidative stress, suggesting a direct role in modulating mitochondrial dynamics .

How can I optimize KRT8 antibody protocols for dual/multiple immunofluorescence staining?

Optimizing multiple immunofluorescence protocols involving KRT8 requires careful consideration of several factors:

• Antibody selection:

  • Choose KRT8 antibodies raised in different host species than other target antibodies

  • Consider using directly conjugated antibodies with non-overlapping fluorophores

  • For KRT8/KRT18 co-staining, use either individual antibodies or verified cocktails

• Fluorophore selection:

  • Avoid blue fluorescent dyes (CF®405S and CF®405M) for low abundance targets due to higher background and lower fluorescence

  • Select fluorophores with minimal spectral overlap: CF®488A (Ex/Em: 490/515nm) for green channel and CF®568 (Ex/Em: 562/583nm) for red channel

• Staining protocol optimization:

  • Sequential rather than simultaneous antibody incubation to minimize cross-reactivity

  • Include appropriate blocking steps with normal serum from the secondary antibody host species

  • Validate antibody specificity with single-stain controls

• Image acquisition considerations:

  • Use sequential scanning to minimize bleed-through

  • Include proper negative controls and single-stained samples for setting acquisition parameters

For detecting KRT8 alongside other cytokeratins or epithelial markers, optimizing antibody concentration is critical - excessive primary antibody can result in non-specific staining or cross-reactivity .

What is the relationship between KRT8 expression and cancer progression?

KRT8 expression has significant implications in cancer biology, particularly in lung adenocarcinoma (LUAD). Research demonstrates that KRT8 is upregulated in LUAD tissues compared to normal tissues, with expression increasing from stage I to stage III .

Key findings on KRT8's role in cancer:

Experimental approaches for studying KRT8 in cancer:

These findings suggest KRT8 could serve as both a biomarker and therapeutic target in lung adenocarcinoma and potentially other epithelial cancers .

How can I troubleshoot inconsistent KRT8 staining patterns across different tissue types?

Inconsistent KRT8 staining across tissues can result from several factors:

• Tissue-specific expression levels:

  • KRT8 expression varies naturally across epithelia (simple vs. stratified)

  • Keratinizing squamous carcinomas are typically negative for KRT8, while adenocarcinomas are positive

• Fixation and processing variables:

  • Optimize fixation time (overfixation can mask epitopes)

  • Use appropriate antigen retrieval methods (heat-induced vs. enzymatic)

  • Consider tissue-specific permeabilization requirements

• Antibody selection factors:

  • Different antibody clones may recognize different epitopes that are variably accessible across tissues

  • Some antibodies perform better in frozen sections while others are optimized for formalin-fixed paraffin-embedded (FFPE) tissues

• Protocol optimization by tissue type:

  • Adjust antibody concentration based on target tissue (1:50 for IHC is standard starting point)

  • Modify incubation times and temperatures

  • Test different blocking reagents to reduce background

For tissues with minimal KRT8 expression (like normal epidermis), more sensitive detection systems may be required. Alternatively, consider using KRT8/18 cocktail antibodies which can enhance detection of simple epithelia and some pathological cells like Paget cells in the epidermis .

What controls should I use when studying KRT8's role in epithelial-to-mesenchymal transition?

When investigating KRT8's role in EMT, comprehensive controls are essential:

• Positive tissue controls:

  • Simple epithelia (colon, lung, prostate) for KRT8 expression

  • Epithelial cancer cell lines with known KRT8 expression (HCC827, H1975)

• Negative tissue controls:

  • Keratinizing squamous carcinomas (typically KRT8-negative)

  • Mesenchymal tissues or fibroblast cell lines

• Experimental controls for KRT8 manipulation studies:

  • Vector-only controls for overexpression studies

  • Non-targeting shRNA for knockdown experiments

  • Isotype controls for antibody specificity validation

• EMT marker validation:

  • Monitor expression of established EMT markers alongside KRT8:

    • Epithelial markers: E-cadherin

    • Mesenchymal markers: N-cadherin, Vimentin, Slug

    • Matrix remodeling enzymes: MMP2

• Pathway activation controls:

  • Positive controls for NF-κB pathway activation (TNF-α treatment)

  • Pathway inhibitor controls (CAPE for NF-κB inhibition)

These controls ensure the observed effects are specifically related to KRT8's function in EMT rather than non-specific experimental artifacts or pathway cross-talk.

How do I differentiate between KRT8 and other cytokeratin family members in my experiments?

Distinguishing between closely related cytokeratins requires careful experimental design:

• Antibody selection strategies:

  • Choose monoclonal antibodies with verified specificity for KRT8

  • Validate antibodies using Western blot against purified cytokeratin standards

  • Consider the specific epitope targeted by the antibody (N-terminal, rod domain, or C-terminal)

• Cross-reactivity testing:

  • Test antibodies on samples with known expression of different cytokeratins

  • Include knockout/knockdown controls to confirm specificity

  • Consider using recombinant cytokeratin proteins as competition controls

• Multiple detection methods:

  • Complement immunodetection with mRNA analysis (qPCR or RNA-seq)

  • Use multiple antibody clones targeting different KRT8 epitopes

  • Employ mass spectrometry for definitive protein identification

• Co-expression analysis:

  • KRT8 typically pairs with KRT18 in simple epithelia

  • Use this pairing pattern to distinguish from other cytokeratin pairs

When studying heteropolymers like KRT8/KRT18, consider using cocktail antibodies specifically designed to recognize both partners simultaneously, which provides more reliable identification of the functional cytokeratin unit in tissues .

How can KRT8 antibodies be utilized in studying autophagy and mitophagy?

KRT8 has recently emerged as an important regulator of autophagy and mitophagy processes. Experimental approaches to investigate this relationship include:

• Co-localization studies:

  • Use KRT8 antibodies with autophagy markers (LC3, p62/SQSTM1)

  • Monitor KRT8's presence at mitochondrial fission sites using fluorescently-tagged KRT8 and mitochondrial markers

  • Track temporal dynamics of KRT8 recruitment during mitophagy induction

• Functional mitophagy assays with KRT8 manipulation:

  • Measure mitophagy flux in KRT8-overexpressing vs. knockdown cells

  • Analyze mitochondrial morphology changes using MitoTracker and live-cell imaging

  • Assess mitochondrial function (membrane potential, ROS production) under oxidative stress

• Mechanistic pathway analysis:

  • Investigate KRT8 interactions with mitophagy machinery proteins

  • Study effects of KRT8 phosphorylation on mitophagy efficiency

  • Examine KRT8-mediated regulation of Golgi resident small GTPase RAB33B trafficking

Research indicates that KRT8 facilitates mitophagy flux, which suppresses the accumulation of damaged mitochondria and consequently diminishes necrotic cell death under oxidative stress conditions . This protective role suggests KRT8 as a potential therapeutic target in conditions involving mitochondrial dysfunction.

What are the considerations for using KRT8 antibodies in different species models?

Working with KRT8 antibodies across different experimental animal models requires attention to several factors:

• Sequence homology and epitope conservation:

  • Human KRT8 shares significant homology with mouse, rat, and monkey orthologs

  • Epitope mapping is essential when transitioning between species models

  • Some antibody clones like UMAB1 demonstrate validated reactivity across human, monkey, mouse, and rat samples

• Species-specific validation protocols:

  • Western blot validation using species-specific tissue lysates

  • Immunohistochemistry on known KRT8-expressing tissues from target species

  • Include knockout/knockdown controls when available

• Application-specific considerations:

  • For xenograft models, select antibodies that distinguish between human and host KRT8

  • In transgenic models, consider antibodies that recognize specific KRT8 mutations or tags

  • For immunoprecipitation studies, validate antibody efficiency in the species of interest

• Cross-reactivity assessment:

  • Test for cross-reactivity with other cytokeratin family members in the target species

  • Validate with appropriate negative controls (tissues known to lack KRT8 expression)

The research demonstrates successful use of KRT8 antibodies in various experimental models, including human cell lines (ARPE-19, HCC827, H1975), mouse models (LLC cells in lung cancer studies), and rat models for intervertebral disc degeneration studies .

How can I design experiments to study KRT8's role in mechanical stress responses?

Recent research has revealed KRT8's involvement in cellular responses to mechanical stress, particularly in intervertebral disc degeneration. To investigate this role:

• In vivo mechanical stress models:

  • Tail compression models in rodents show KRT8 expression changes under mechanical load

  • Conditional knockout of Krt8 in nucleus pulposus (NP) cells aggravates load-induced intervertebral disc degeneration

• In vitro compression studies:

  • Apply mechanical compression to primary cells with different KRT8 expression levels

  • Monitor time-dependent changes in KRT8 expression (transcription, translation, degradation)

  • Use precise pressure systems (e.g., 1.0 MPa compression for 24-48h)

• Molecular pathway analysis:

  • Investigate RHOA-PKN pathway activation in response to mechanical load

  • Study KRT8 phosphorylation status (particularly Ser43) under different loading conditions

  • Analyze proteasome involvement in KRT8 degradation using inhibitors like MG132

• Experimental design considerations:

  • Include time course analysis (KRT8 shows distinct expression patterns at different time points)

  • Combine overexpression and knockdown approaches

  • Use translation inhibitors (cycloheximide) and proteasome inhibitors (MG132) to distinguish between synthesis and degradation effects

The research indicates that KRT8 expression initially increases under mechanical stress (up to 36 hours) followed by a significant drop, suggesting complex transcriptional and post-translational regulation mechanisms that can be targeted therapeutically .

What emerging technologies are enhancing KRT8 antibody applications in research?

Several cutting-edge technologies are expanding the utility of KRT8 antibodies in research:

• Advanced imaging technologies:

  • Super-resolution microscopy for detailed visualization of KRT8 filament organization

  • Live-cell imaging with GFPSpark-tagged KRT8 for dynamic studies of filament assembly/disassembly

  • Correlative light and electron microscopy (CLEM) for ultrastructural context

• Multiplexed detection systems:

  • Multi-color immunofluorescence with spectral unmixing

  • Cyclic immunofluorescence (CycIF) for simultaneous detection of multiple markers

  • Mass cytometry (CyTOF) for high-dimensional protein analysis

• Conjugation chemistry advancements:

  • CF® dyes offering exceptional brightness and photostability

  • Site-specific conjugation technologies for optimal antibody performance

  • Quantum dot conjugates for enhanced sensitivity and photostability

• High-throughput applications:

  • Tissue microarray analysis for KRT8 expression in large sample cohorts

  • Automated image analysis algorithms for quantitative assessment of KRT8 staining

  • Single-cell techniques for heterogeneity analysis in complex tissues

• Proximity labeling approaches:

  • BioID or APEX2 fusion with KRT8 to identify proximity interactors

  • Visualization of KRT8 interaction networks using proximity ligation assays

These technologies enable researchers to address increasingly complex questions about KRT8's dynamic behavior and functional interactions in normal and pathological contexts .

What are the most promising therapeutic applications targeting KRT8?

Based on current research, several promising therapeutic directions involving KRT8 are emerging:

• Cancer therapeutics:

  • KRT8 shows potential as a biomarker for lung adenocarcinoma and other cancers

  • KRT8 knockdown inhibits proliferation, migration, and invasion of cancer cells

  • Targeting KRT8-regulated EMT pathways may reduce metastasis

• Protection against oxidative stress:

  • KRT8 facilitates mitophagy and protects against necrotic cell death

  • Therapeutic modulation of KRT8 may protect cells from oxidative damage

  • Potential applications in retinal and neurodegenerative diseases

• Intervertebral disc degeneration therapy:

  • Overexpression of KRT8 at early stages of disc degeneration shows protective effects

  • Targeting PKN1 and PKN2 (which phosphorylate KRT8) may be beneficial in late-stage disease

  • Combined approaches modulating both KRT8 and its regulatory pathways may provide therapeutic synergy

Future therapeutic strategies may involve:

• Small molecule inhibitors of KRT8 phosphorylation

• Gene therapy approaches to modulate KRT8 expression

• Targeted delivery systems for KRT8-modulating compounds to specific tissues