KRT35 Antibody

What is KRT35 and why is it important in research?

KRT35 (Keratin 35) is a type I cuticular Ha5 protein that belongs to the keratin gene family. As an acidic protein, it heterodimerizes with type II keratins to form hair and nails . KRT35 is primarily expressed in hair follicles, particularly in supramatricial cells and lowermost cortical cells of the hair bulb . Recent evolutionary and comparative genomics research has identified KRT35 as potentially deregulated across several cancer types, suggesting it participates in protein-protein interaction networks relevant to cancer biology . This makes KRT35 not only important for hair biology studies but also potentially significant in cancer research.

What types of KRT35 antibodies are available for research applications?

Multiple types of KRT35 antibodies are available for research, primarily rabbit polyclonal antibodies with different specificities and conjugations:

Most of these antibodies have been protein G purified to >95% purity .

What are the recommended applications for KRT35 antibodies?

KRT35 antibodies are primarily used in these applications:

• Western Blotting (WB): Most KRT35 antibodies are validated for WB with recommended dilutions ranging from 1:1000 to 1:5000 .

• Immunohistochemistry (IHC): Several antibodies are suitable for IHC with recommended dilutions of 1:20 to 1:200 .

• ELISA: Multiple antibodies, including specialized conjugated versions (Biotin, HRP), are validated for ELISA applications .

The choice of antibody depends on the specific research question, target epitope of interest, and experimental system. Antibodies targeting different regions (N-terminal, C-terminal, or internal domains) may yield different results based on protein conformation, processing, or interactions in the experimental context.

How should I optimize Western blotting conditions for KRT35 detection?

For optimal Western blotting detection of KRT35:

• Sample preparation: Use appropriate lysis buffers that effectively solubilize keratins (containing SDS or urea).

• Protein loading: Load 20-50 μg of total protein per lane.

• Gel percentage: Use 10-12% SDS-PAGE gels as the calculated molecular weight of KRT35 is 50,361 Da .

• Transfer conditions: Transfer proteins to PVDF or nitrocellulose membranes using standard protocols.

• Blocking: Block with 5% non-fat dry milk or BSA in TBST.

• Primary antibody incubation: Dilute primary antibody (1:1000-1:5000) in blocking buffer and incubate overnight at 4°C .

• Detection: Use appropriate secondary antibodies and detection methods based on experimental requirements.

Always include appropriate positive controls (tissues known to express KRT35, such as hair follicle samples) and negative controls.

What is the recommended protocol for immunohistochemistry using KRT35 antibodies?

For IHC applications with KRT35 antibodies:

• Fixation: Fix tissues in 10% neutral buffered formalin.

• Embedding and sectioning: Embed in paraffin and section at 4-6 μm thickness.

• Antigen retrieval: Perform heat-induced epitope retrieval in citrate buffer (pH 6.0) or EDTA buffer (pH 9.0).

• Blocking: Block endogenous peroxidase with 3% H₂O₂ and non-specific binding with serum.

• Primary antibody: Dilute KRT35 antibody at 1:20-1:200 and incubate for 1-2 hours at room temperature or overnight at 4°C .

• Detection: Use appropriate detection systems (HRP/DAB for chromogenic detection).

• Counterstaining: Counterstain with hematoxylin for nuclear visualization.

Since KRT35 is primarily expressed in hair follicles, scalp or skin tissues containing hair follicles serve as positive controls.

How can I determine the specificity of my KRT35 antibody?

Confirming antibody specificity is crucial for reliable research outcomes. Multiple approaches should be used:

• Knockdown/knockout validation: Use siRNA, shRNA, or CRISPR-Cas9 to reduce or eliminate KRT35 expression, then compare antibody signal between control and KRT35-depleted samples.

• Peptide competition assay: Pre-incubate the antibody with the immunizing peptide before application to samples. A specific antibody will show diminished signal.

• Multiple antibody comparison: Use antibodies targeting different epitopes of KRT35 (e.g., N-terminal vs. C-terminal) and compare staining patterns.

• Cross-reactivity testing: Test the antibody on samples from different species to confirm specificity based on the claimed reactivity (human, mouse, etc.).

• Molecular weight verification: Confirm that the detected band in Western blot corresponds to the predicted molecular weight of KRT35 (~50 kDa) .

Remember that KRT35 is part of a large keratin family with potential sequence similarities, so careful validation is essential to ensure specificity.

What tissues or cell lines are recommended as positive controls for KRT35 antibody validation?

Based on the expression profile of KRT35:

• Tissue controls:

  • Human scalp sections with hair follicles (particularly hair bulb regions)

  • Hair shafts

  • Skin samples containing anagen phase hair follicles

• Cell lines:

  • Human follicular keratinocyte cell lines

  • Human hair follicle dermal papilla cells

  • Some cancer cell lines that show upregulation of KRT35 based on recent research

KRT35 shows early expression in the hair follicle, mainly in supramatricial cells and lowermost cortical cells of the hair bulb , making these regions ideal for positive control validation.

What are the most common issues when using KRT35 antibodies and how can they be resolved?

Common issues and solutions include:

• High background in IHC/ICC:

  • Increase blocking time/concentration

  • Optimize antibody dilution (try more dilute solutions)

  • Ensure thorough washing steps

  • Use more specific secondary antibodies

• No signal in Western blot:

  • Verify sample preparation (keratins require proper solubilization)

  • Check if the epitope is masked or degraded

  • Increase protein loading or antibody concentration

  • Try alternative antibodies targeting different epitopes

  • Verify target expression in your sample type

• Multiple bands in Western blot:

  • Could indicate splice variants, post-translational modifications, or degradation products

  • Optimize gel percentage and running conditions

  • Try reducing sample preparation temperature to minimize degradation

  • Cross-check with antibodies targeting different epitopes

• Inconsistent results between applications:

  • Some epitopes may be accessible in certain applications but masked in others

  • Consider using antibodies specifically validated for your application of interest

  • Optimize fixation/preparation conditions for each application

How should KRT35 antibodies be stored to maintain optimal performance?

For optimal storage and handling of KRT35 antibodies:

• Long-term storage: Store antibodies at -20°C in small aliquots to prevent repeated freeze-thaw cycles .

• Working stocks: Keep at 4°C for up to 2 weeks .

• Aliquoting: Upon first use, prepare small working aliquots to avoid repeated freeze-thaw cycles that can degrade antibody performance.

• Buffer considerations: Most KRT35 antibodies are supplied in PBS with preservatives like 0.03% Proclin 300 or 0.09% sodium azide, and 50% glycerol .

• Handling precautions: Note that preservatives like ProClin are hazardous substances requiring proper handling .

• Expiration guidelines: Generally follow manufacturer recommendations, but antibody performance should be validated regularly regardless of expiration dates.

What is the current understanding of KRT35's role in cancer research?

Recent systems biology approaches, including comparative genomics and phylogenetics, have identified KRT35 among the keratin genes that are deregulated across several cancer types . Findings indicate:

• KRT35 participates in protein-protein interaction networks common to several cancer types.

• Phylogenetically conserved keratin genes, potentially including KRT35, might have been under similar evolutionary pressure in cancer-bearing species.

• Text mining analyses have detected semantic relations between keratins (including KRT35) and cancer.

• The evolutionary history of keratins has been reconstructed in the context of cancer, suggesting potential roles in cancer biology.

This emerging research suggests KRT35 may have broader significance beyond its structural role in hair formation, potentially serving as a biomarker or functional contributor in certain cancer types .

How do KRT35 antibodies compare in cross-species reactivity for comparative biology studies?

The cross-species reactivity of KRT35 antibodies varies significantly, which is crucial for researchers conducting comparative biology or using model organisms:

When designing comparative studies across species, researchers should carefully select antibodies with validated cross-reactivity for their species of interest. The antibody targeting AA 201-250 appears to have the broadest cross-species reactivity and may be most suitable for comparative studies .

What bioinformatic resources are available for analyzing KRT35 in research contexts?

Several bioinformatic resources are available for KRT35 analysis:

• Database accessions:

  • UniProt: Primary accession Q92764; Secondary accessions O76012, Q92651; Entry name KRT35_HUMAN

  • NCBI Gene ID: 3886

  • OMIM: 602764

  • KEGG: hsa:3886

  • String: 9606.ENSP00000377558

  • Ensembl: ENST00000393989, ENSP00000377558, ENSG00000197079

• Knowledge resources:

  • Pharos database provides knowledge scores for KRT35, with highest values for gene perturbation (0.98), interacting protein (0.71), and protein domain (0.58) .

  • PubMed score: 124.74, with 2 Gene RIFs and 126 available antibodies .

• Comparative genomics:

  • Cross-genome search tools using KRT35 protein sequences can identify orthologs in model organisms including mouse, cow, chicken, frog, and zebrafish .

These resources enable researchers to perform comprehensive analyses of KRT35 structure, function, and evolutionary relationships.

What are the key considerations for using KRT35 antibodies in cancer tissue microarrays or biomarker studies?

When using KRT35 antibodies in cancer tissue microarrays (TMAs) or biomarker studies:

• Antibody selection:

  • Choose antibodies with high specificity and sensitivity for human tissues

  • Consider using antibodies targeting conserved epitopes if comparing across cancer types

• Controls:

  • Include both positive controls (hair follicle-containing tissues) and negative controls

  • Use normal adjacent tissue controls whenever possible

  • Consider including gradient standards if performing quantitative analysis

• Scoring systems:

  • Develop and validate a scoring system appropriate for KRT35 expression patterns

  • Consider both intensity and extent of staining

  • Use digital pathology tools for quantitative assessment when possible

• Interpretation challenges:

  • Be aware that keratin expression can be affected by tissue fixation and processing

  • Consider the heterogeneity of KRT35 expression within tumors

  • Correlate with other biomarkers and clinical data for meaningful interpretation

• Validation approach:

  • Validate findings with orthogonal methods (e.g., qRT-PCR, RNA-seq)

  • Consider multi-institutional validation cohorts

  • Follow REMARK guidelines for biomarker studies