PTK6 Antibody

What is PTK6 and why is it important in research?

PTK6 (Protein Tyrosine Kinase 6), also known as BRK (Breast tumor Kinase), is a cytoplasmic nonreceptor protein kinase that functions as an intracellular signal transducer predominantly in epithelial tissues. PTK6 belongs to a distinct family of nonreceptor tyrosine kinases that are distantly related to Src kinases .

PTK6 has emerged as a significant research target because:

• It is aberrantly overexpressed in multiple cancer types including breast cancer (up to 86% of cases), bladder cancer, colorectal cancer, and kidney renal clear cell carcinoma (KIRC)

• Its expression correlates with cancer progression, metastasis, and poor patient outcomes in multiple cancer types

• It represents a potential therapeutic target and biomarker for cancer diagnosis and prognosis

What applications can PTK6 antibodies be used for?

PTK6 antibodies have been validated for multiple research applications as detailed in the following table:

Note: It is recommended to titrate the antibody in each testing system to obtain optimal results as outcomes may be sample-dependent .

How should I prepare samples for PTK6 detection by immunohistochemistry?

For optimal PTK6 detection in tissue samples by IHC:

• Fixation: Formalin fixation followed by paraffin embedding is commonly used

• Sectioning: 4-5 μm thick sections are typically prepared

• Antigen retrieval: Two recommended methods:

  • Primary method: TE buffer at pH 9.0

  • Alternative method: Citrate buffer at pH 6.0

• Blocking: Use appropriate blocking solution (e.g., PBS with BSA)

• Primary antibody incubation: Dilute PTK6 antibody 1:20-1:200 and incubate appropriately (overnight at 4°C shows good results)

• Detection system: Typically DAB-based detection systems work well

• Counterstaining: Hematoxylin for nucleus visualization

According to data from clinical studies, PTK6 antibodies have been successfully used at 1:400 dilution with overnight incubation at 4°C for reliable detection in breast cancer tissues .

How do I determine the appropriate positive and negative controls for PTK6 antibody experiments?

Positive Controls:

• Cell lines with confirmed PTK6 expression: HeLa, MCF-7, T24, and EJ cells have demonstrated reliable PTK6 expression

• Tissue samples: Colon cancer tissues, breast cancer tissues, and spleen tissue from mouse/rat have been validated for PTK6 expression

• Recombinant systems: Cells transfected with PTK6-expressing constructs such as Myc-tagged full-length human wild-type (WT) PTK6

Negative Controls:

• Knockdown validation: Cells with stable PTK6 knockdown using verified shRNAs (e.g., TCRN0000021549 and TCRN0000021552 from the PTK6 Mission TCR shRNA Target Set)

• Antibody controls: IgG isotype controls matching the host species of the PTK6 antibody

• Peptide blocking: Pre-incubation of antibody with immunizing peptide to confirm specificity

• Normal tissue: Some normal tissues express low or undetectable levels of PTK6 and can serve as relative negative controls

Technical validation approaches:

• Compare staining with multiple PTK6 antibodies targeting different epitopes

• Include secondary antibody-only controls to rule out non-specific binding

• Use antibody concentration gradients to establish signal-to-noise ratio thresholds

How can I detect different phosphorylation states of PTK6?

PTK6 function is regulated by phosphorylation at specific residues, and phospho-specific antibodies can be used to distinguish active versus inactive forms:

• Active PTK6 detection:

  • Use antibodies specific to phospho-Tyrosine 342 (PY342)

  • These antibodies (e.g., 09-144 from EMD Millipore) detect the catalytically active form of PTK6

  • Recommended dilution: Follow manufacturer guidelines, typically 1:500-1:1000 for Western blot

• Inactive PTK6 detection:

  • Use antibodies specific to phospho-Tyrosine 447 (PY447)

  • These antibodies (e.g., ab138368 from Abcam) detect the auto-inhibited form of PTK6

  • This phosphorylation creates a conformation that inhibits kinase activity

• Total PTK6 detection:

  • Use antibodies recognizing PTK6 regardless of phosphorylation state

  • These provide baseline information on total protein expression

For studying PTK6 activation mechanisms, researchers can use recombinant constructs with specific mutations:

• PTK6-WT: Wild-type protein exhibiting normal regulation

• PTK6-YF: Constitutively active mutant (Y447F mutation)

• PTK6-KM: Kinase-defective mutant

What methods can I use to quantify PTK6 expression levels in patient samples?

Several validated methods for quantifying PTK6 expression in patient samples include:

1. Immunohistochemistry (IHC) scoring:

• Semi-quantitative scoring based on staining intensity and percentage of positive cells

• Common scoring systems include:

  • 0: Negative staining

  • 1+: Weak staining

  • 2+: Moderate staining

  • 3+: Strong staining

• Percentage of positive cells typically categorized as 0-25%, 26-50%, 51-75%, or >75%

• Final score often calculated as intensity × percentage for stratification

2. Quantitative PCR (qPCR):

• Real-time PCR using primers specific for PTK6 transcripts

• Amplifluor technology with PCR primers designed using Beacon Designer software has shown reliable results

• Normalization against housekeeping genes such as cytokeratin 19 (CK19)

• Conditions: 94°C for 10 min followed by 55 cycles of 94°C for 10s, 55°C for 30s, and 72°C for 15s

3. Western blot analysis with densitometry:

• Semi-quantitative analysis of protein levels

• Densitometry analysis using ImageJ software with normalization to loading controls such as GAPDH or β-Actin

• Sample preparation using NP-40 lysis buffer with protease and phosphatase inhibitors

4. Digital pathology approaches:

• Whole slide imaging followed by quantitative image analysis

• Algorithm-based quantification of staining intensity and distribution

• Allows for larger sample analysis and reduced inter-observer variability

How does PTK6 expression correlate with cancer progression and patient outcomes?

PTK6 expression has been extensively studied in relation to cancer progression and outcomes across multiple cancer types:

Breast Cancer:

• Overexpression in up to 86% of breast cancer cases

• PTK6 transcript expression has prognostic significance with higher levels associated with adverse outcomes independent of nodal status

• ER+ and Her2+ subtypes express the highest levels of PTK6 transcript

• The ratio of PTK6 to its alternate form (ALT-PTK6) is significantly different between ER+ and ER- tumors (p=0.042)

Kidney Renal Clear Cell Carcinoma (KIRC):

Bladder Cancer:

• Significantly higher expression in bladder cancer tissues compared to normal controls

• Overexpression significantly correlates with:

  • T classification (tumor invasion depth)

  • N classification (lymph node involvement)

  • Higher grade tumors

  • Disease recurrence

  • Poorer clinical prognosis

Colorectal Cancer (CRC):

• Aberrantly elevated in tumor tissues compared to adjacent normal tissues (55.6% vs 34.5%)

• High PTK6 expression correlates with poor prognosis

• Expression increases with advancing stage (stage II-IV vs stage 0-I)

• Linked to chemoresistance in CRC patients

What mechanisms explain how PTK6 promotes cancer progression?

Research has revealed several mechanisms by which PTK6 contributes to cancer development and progression:

1. Regulation of Epithelial-Mesenchymal Transition (EMT):

• PTK6 knockdown in SW480 colon cancer cells reduces expression of epithelial markers E-Cadherin and ZO-1

• Simultaneously increases expression of mesenchymal markers ZEB-1, Vimentin, and Claudin-1

• Similar effects observed at mRNA level with decreased CDH1 (E-cadherin) and increased ZEB1, VIM, and dramatic increases in TWIST1

• These changes promote cancer cell invasion and metastasis

2. Inhibition of Apoptosis:

• PTK6 regulates the expression of Bim, a pro-apoptotic Bcl2 family member

• Downregulation of PTK6 enhances Bim expression, resulting in apoptotic cell death in Her2+ breast cancer cells

• PTK6 inhibition impairs growth in 3-D Matrigel cultures and inhibits growth of Her2+ primary tumor xenografts

• Regulation of Bim occurs via p38 MAPK pathway activation rather than through Erk/MAPK or Akt signaling

3. Immune Evasion:

• PTK6 positively correlates with immune checkpoint molecules including:

  • CD276 (B7-H3)

  • TGFB1

  • EDNRB

  • SLAMF7

  • CTLA4

  • TIGIT

  • LAG3

  • PDCD1

  • IL13

• Also associated with biomarkers relevant to immunotherapy response:

  • Tumor Mutation Burden (TMB)

  • Microsatellite Instability (MSI)

  • Neo-antigen (NEO)

  • DNA Ploidy changes

4. Promotion of Cancer Stemness and Chemoresistance:

• PTK6 contributes to chemoresistance in colorectal cancer

• Inhibition of PTK6 with small molecule inhibitor XMU-MP-2 increases sensitivity to chemotherapeutic agents (5-FU/L-OHP)

• Regulates JAK2/STAT3 signaling pathway implicated in cancer stem cell maintenance

How can PTK6 antibodies be used to evaluate potential therapeutic approaches?

PTK6 antibodies provide critical tools for evaluating therapeutic approaches targeting PTK6 or related pathways:

1. Monitoring target inhibition:

• PTK6 antibodies can be used to assess the efficacy of PTK6-targeting drugs

• Phospho-specific antibodies (detecting Y342 or Y447) help determine whether inhibitors effectively block PTK6 activation

• Western blot and IHC analysis with these antibodies provide complementary information about drug effects on PTK6 at protein level

2. Validating genetic knockdown approaches:

• Antibodies confirm successful PTK6 protein reduction following siRNA or shRNA treatment

• Functional studies examining cancer cell growth, migration, and apoptosis after PTK6 knockdown can be validated with antibodies

3. Patient stratification for clinical trials:

• IHC with PTK6 antibodies can identify patients with high PTK6 expression who might benefit from PTK6-targeted therapies

• Multiple studies show correlation between PTK6 expression and poor prognosis, suggesting potential therapeutic value in PTK6 inhibition

4. Studying resistance mechanisms:

• PTK6 plays a role in resistance to targeted therapies like Lapatinib in Her2+ breast cancer

• Antibodies help track changes in PTK6 expression and activation in treatment-resistant cells

• Research shows PTK6 inhibition promotes apoptosis in Lapatinib-resistant Her2+ breast cancer cells by enhancing Bim expression

5. Biomarker development:

• PTK6 antibodies aid in developing PTK6 as a biomarker for:

  • Prognosis prediction

  • Therapy response prediction

  • Patient selection for immunotherapy (given correlations with immune checkpoints)

How do subcellular localization patterns of PTK6 affect its function in cancer?

PTK6 subcellular localization significantly influences its biological functions, with different compartmental distribution associated with distinct cancer phenotypes:

Cytoplasmic vs. Nuclear Localization:

• PTK6 has been observed in both cytoplasmic and nuclear compartments of cancer cells

• Immunofluorescence staining shows PTK6 localization in both compartments in colorectal cancer cell lines

• Research suggests that subcellular redistribution of PTK6 can alter its role from tumor suppressive to oncogenic

Methodological considerations for studying localization:

• Immunofluorescence approaches:

  • Use well-validated antibodies at 1:50-1:500 dilution

  • Include nuclear counterstains (DAPI/Hoechst) for clear delineation of compartments

  • Consider confocal microscopy for accurate subcellular resolution

• Subcellular fractionation:

  • Western blot analysis of nuclear and cytoplasmic fractions

  • Requires careful validation of fractionation quality with compartment-specific markers

  • Compare total PTK6 levels with active/inactive (phosphorylated) forms in each compartment

• Proximity ligation assays:

  • Identify in situ protein-protein interactions in specific cellular compartments

  • Valuable for characterizing PTK6 signaling partners in different cellular locations

What approaches are recommended for studying PTK6 protein-protein interactions?

Investigating PTK6 interactions with signaling partners provides critical insights into its oncogenic mechanisms. These approaches are recommended:

1. Co-immunoprecipitation (Co-IP):

• Validated protocol:

  • Prepare cell lysates in NP-40 lysis buffer with protease and phosphatase inhibitors

  • Pre-clear lysate with 50% slurry beads (equilibrated with IP buffer) for 30 minutes

  • Incubate with PTK6 antibody (e.g., C18) for 2 hours

  • Add 35 μl of 50% slurry beads for 1 hour at 4°C on rotation

  • Wash beads three times with IP wash buffer

  • Elute by boiling with 2× SDS sample buffer at 95°C for 3 minutes

  • Analyze by Western blotting

2. Protein-protein interaction (PPI) network analysis:

• Tools like STRING (https://string-db.org/) can identify top PTK6-interacting proteins

• Analysis of PTK6-related genes and PTK6-interaction genes can be performed using KEGG pathway and Gene Ontology (GO) enrichment

3. Proximity-dependent biotin identification (BioID):

• Fusion of PTK6 with a biotin ligase

• Allows identification of proximal proteins in living cells

• Particularly useful for detecting weak or transient interactions

4. PTK6 mutant constructs for mechanistic studies:

• Researchers can use:

  • Myc-tagged full-length human wild-type (WT) PTK6

  • Constitutively active (YF) mutant

  • Kinase-defective (KM) mutant

• These constructs help distinguish between kinase-dependent and independent functions

What considerations are important when comparing PTK6 expression data across different studies and platforms?

Researchers should be aware of several factors that may affect comparability of PTK6 expression data:

1. Antibody selection and validation:

• Different antibodies target distinct epitopes of PTK6

• Phospho-specific antibodies (e.g., PY342, PY447) detect different activation states

• Antibody validation methods vary between studies

• Recommendation: Review antibody validation data including controls used (knockout/knockdown, peptide competition)

2. Detection methods and quantification:

• IHC scoring systems vary (H-score, Allred score, percentage positive)

• qPCR normalization approaches differ (various housekeeping genes used)

• Western blot quantification methods may include different loading controls

• Recommendation: Standardize to established protocols when possible or adjust for methodological differences

3. Sample processing variations:

• Fixation methods affect antigen preservation

• Antigen retrieval protocols influence epitope accessibility

• RNA/protein extraction methods impact yield and quality

• Recommendation: Note fixation and processing details when comparing studies

4. Alternative splicing and isoforms:

• PTK6 transcript levels can be affected by alternative splicing

• The PTK6/ALT-PTK6 ratio shows significant differences between ER+ and ER- tumors (p=0.042)

• Recommendation: Ensure primers or antibodies detect the relevant isoforms

5. Data normalization across platforms:

• Integration of data from different sources (TCGA, GEO, GTEx) requires normalization

• Log2 transformation of transcripts per million reads (TPM) is commonly used (Log2(TPM+1))

• Recommendation: Apply appropriate statistical methods for cross-platform normalization

What are common issues with PTK6 antibodies and how can they be addressed?

Researchers may encounter several challenges when working with PTK6 antibodies. Here are common issues and recommended solutions:

1. Weak or no signal:

• Potential causes:

  • Insufficient antigen

  • Epitope masking during fixation

  • Degraded antibody

  • Suboptimal antibody concentration

• Solutions:

  • Verify PTK6 expression in positive controls

  • Optimize antigen retrieval (try both TE buffer pH 9.0 and citrate buffer pH 6.0)

  • Test different antibody concentrations within recommended range (1:20-1:200 for IHC)

  • Use fresh antibody aliquots stored according to manufacturer recommendations (-20°C, with glycerol)

2. High background:

• Potential causes:

  • Insufficient blocking

  • Excessive antibody concentration

  • Non-specific binding

  • Cross-reactivity

• Solutions:

  • Increase blocking time/concentration

  • Dilute antibody further

  • Include additional washing steps

  • Pre-absorb antibody with relevant species proteins

  • Use IgG isotype controls to assess non-specific binding

3. Inconsistent results:

• Potential causes:

  • Batch-to-batch antibody variability

  • Inconsistent sample processing

  • Variation in fixation times

• Solutions:

  • Validate each new antibody lot against previous results

  • Standardize fixation and processing protocols

  • Include consistent positive and negative controls with each experiment

  • Consider pooling antibody aliquots for long-term studies

4. Issues specific to phospho-PTK6 detection:

• Potential causes:

  • Rapid dephosphorylation during sample preparation

  • Phosphatase activity

  • Epitope masking

• Solutions:

  • Include phosphatase inhibitors in all buffers

  • Process samples rapidly and maintain cold temperatures

  • Test multiple antigen retrieval methods

  • Consider specialized fixatives that better preserve phospho-epitopes

How should researchers validate PTK6 antibody specificity for their experimental systems?

Thorough validation of PTK6 antibodies is essential for reliable research results. Recommended validation approaches include:

1. Genetic validation:

• Use PTK6 knockdown/knockout systems:

  • shRNA approaches (validated shRNAs: TCRN0000021549, TCRN0000021552)

  • CRISPR-Cas9 knockout models

  • Compare antibody signal between PTK6-expressing and PTK6-depleted samples

  • Confirm knockdown efficiency by qPCR in parallel

2. Expression system validation:

• Overexpress tagged PTK6 constructs:

  • Compare antibody signal with anti-tag antibody detection

  • Use PTK6 mutant constructs (WT, YF, KM) to validate specificity

  • Test signal in cells with naturally low PTK6 expression (e.g., normal colon epithelial NCM460 cells)

3. Western blot validation:

• Confirm single band at expected molecular weight (52 kDa)

• Test multiple antibodies targeting different epitopes

• Include positive controls (HeLa cells, MCF-7 cells, spleen tissue)

• Peptide competition with immunizing antigen to confirm specificity

4. Cross-application validation:

• Compare results across multiple applications (WB, IHC, IF)

• Consistency across applications increases confidence in specificity

• Different applications may require different antibody concentrations:

  • WB: 1:500-1:2000

  • IHC: 1:20-1:200

  • IF/ICC: 1:50-1:500

5. Cross-species reactivity assessment:

• Confirmed reactivity with human, mouse, and rat samples

• Test conservation of target epitope across species of interest

• Validate antibody in each species separately

What are the best practices for optimizing PTK6 immunohistochemistry protocols?

For optimal PTK6 detection by immunohistochemistry, consider these evidence-based recommendations:

1. Sample preparation:

• Fixation: 10% neutral buffered formalin for 24-48 hours

• Processing: Standard paraffin embedding protocols

• Sectioning: 4-5 μm sections on positively charged slides

• Storage: Use freshly cut sections when possible; stored sections should be used within 4 weeks

2. Antigen retrieval optimization:

• Primary recommended method: TE buffer at pH 9.0

• Alternative method: Citrate buffer at pH 6.0

• Duration: 10-20 minutes at high pressure or microwave heating

• Cooling: Allow slow cooling to room temperature (20 minutes)

• Comparative testing: Side-by-side comparison of different methods may be necessary for optimal results

3. Blocking and antibody incubation:

• Blocking: 5-10% normal serum (matching secondary antibody species) with 1% BSA

• Primary antibody dilution range: 1:20-1:200 for IHC

• Systematic titration: Test multiple dilutions on positive control tissues

• Incubation conditions: 4°C overnight incubation has shown good results in clinical studies

• Secondary antibody: Match to primary antibody host species (typically anti-rabbit)

4. Detection and signal development:

• DAB development: Monitor carefully to balance signal and background

• Counterstaining: Light hematoxylin to visualize tissue context without obscuring PTK6 signal

• Mounting: Use appropriate mounting media compatible with your detection system

5. Quality control measures:

• Run parallel positive controls (colon cancer tissue shows reliable PTK6 expression)

• Include negative controls (antibody diluent only, isotype control)

• Process all samples in a batch under identical conditions

• Document all protocol parameters for reproducibility