HCK Antibody

What is HCK and why is it important in research?

HCK (hematopoietic cell kinase) is a member of the SRC family of cytoplasmic tyrosine kinases expressed primarily in hematopoietic cells of myeloid and B-lymphocyte lineages. It plays crucial roles in cell signaling pathways related to immune responses, inflammation, and cancer development. HCK has emerged as an important research target because excessive HCK activation is associated with several types of leukemia and solid malignancies, including breast and colon cancer, where it correlates with decreased patient survival rates . Furthermore, HCK enhances secretion of growth factors and pro-inflammatory cytokines from myeloid cells and promotes macrophage polarization toward a tumor-promoting phenotype, making it a valuable target for both basic research and therapeutic development .

What are the structural characteristics of the HCK protein that researchers should be aware of?

HCK exists in multiple isoforms, with the primary protein reported to be approximately 59.6 kilodaltons in mass . Researchers should be aware that alternate translation initiation site usage, including a non-AUG (CUG) codon, results in the production of two distinct isoforms: p59Hck and p61Hck . These isoforms demonstrate different subcellular localization patterns, which is critical knowledge when designing immunofluorescence or subcellular fractionation experiments. The protein contains a catalytic domain and regulatory regions that control its activation state. Most importantly for antibody-based research, HCK contains specific phosphorylation sites, particularly Tyr411 (pTyr411), which serves as a critical marker of HCK activation and can be detected using phospho-specific antibodies in techniques such as Phosflow analysis .

How should researchers select the most appropriate HCK antibody for their specific experimental applications?

When selecting an HCK antibody, researchers should consider several critical factors:

• Target epitope: Determine whether you need an antibody targeting the N-terminal region (for detecting all isoforms), C-terminal region, or a specific phosphorylation site like pTyr411 for activation studies .

• Application compatibility: Verify the antibody has been validated for your specific application. For example, search results show antibodies validated for Western blot (WB), immunohistochemistry (IHC-p), immunofluorescence (IF), immunoprecipitation (IP), ELISA, and flow cytometry .

• Species reactivity: Confirm cross-reactivity with your experimental species. Available antibodies show reactivity with human, mouse, rat, and sometimes additional species like rabbit and dog .

• Clonality: Choose between monoclonal antibodies for high specificity to a single epitope (like the mouse monoclonal IgG1 from Santa Cruz Biotechnology) or polyclonal antibodies for broader epitope recognition .

• Validation data: Review the supplier's validation data, including published citations, figures showing expected bands in Western blots, or appropriate staining patterns in IHC/IF applications .

What validation steps should be performed to ensure HCK antibody specificity?

To ensure antibody specificity, researchers should implement the following validation steps:

• Positive and negative controls: Use cell lines with known HCK expression levels (high in myeloid lineage cells) versus those with minimal expression.

• Knockdown verification: Implement HCK-specific siRNA or antisense oligonucleotides to confirm signal reduction. As reported in the literature, antisense ODN sequences like 5′-GAA CTT GGA CTT CAT GCA CCC-3′ have been successfully used to inhibit endogenous HCK expression .

• Phospho-specificity testing: For phospho-specific antibodies, treat samples with phosphatase to verify signal loss.

• Cross-reactivity assessment: Test against related SRC family kinases to ensure specificity for HCK.

• Comparison across techniques: Validate findings using multiple methods (e.g., Western blot plus immunofluorescence).

• Recombinant protein: Use purified recombinant HCK protein as a positive control for antibody testing.

What are the optimal protocols for using HCK antibodies in flow cytometry?

For optimal flow cytometry with HCK antibodies, follow these methodological guidelines:

• Cell preparation and fixation:

  • Harvest 1 × 10^6 cells per sample

  • Fix cells with BD Phosflow Fix Buffer I at 37°C for 10 minutes

  • Centrifuge at 300g for 5 minutes and wash twice with Phosflow Perm/Wash Buffer I

• Staining procedure:

  • For cell lines: Stain with HCK (pTyr 411) antibody (or other HCK antibodies)

  • For primary cells: Co-stain with lineage markers (e.g., CD20–APC-Cy7 for B cells)

  • Incubate in the dark for 30 minutes at room temperature

  • Wash three times with BD Phosflow Perm/Wash Buffer I

  • Apply secondary antibody (e.g., anti-rabbit IgG DyLight-649) if using unconjugated primary antibodies

  • Incubate for an additional 20 minutes and wash twice

• Data acquisition and analysis:

  • Acquire minimum of 10,000 events using a flow cytometer (e.g., BD FACSCanto II)

  • For primary samples, gate on the appropriate lineage marker before analyzing HCK expression

  • Compare to appropriate isotype controls to determine positive staining

How can researchers effectively use HCK antibodies in the context of studying cancer pathways?

To effectively study HCK in cancer pathways, researchers should implement these methodological approaches:

• Multi-parameter signaling analysis:

  • Use phospho-specific HCK antibodies (pTyr411) alongside other pathway markers (e.g., pBTK, pERK, pAKT) to map signaling networks

  • Implement Phosflow or multiplex Western blotting to assess pathway activation following treatment with inhibitors

• HCK activity assessment in patient samples:

  • For blood cancers like Waldenström macroglobulinemia (WM) or acute myeloid leukemia, use CD19 or other lineage markers with HCK antibodies to analyze malignant populations

  • Compare HCK activation levels between healthy donor cells and patient samples to establish baseline differences

• Inhibitor efficacy studies:

  • Use HCK antibodies in cell viability assays following HCK inhibitor treatment

  • Perform target engagement studies using pull-down experiments with biotinylated inhibitors to confirm binding to HCK

  • Assess downstream signaling changes with multi-parameter analysis

• Genetic manipulation approaches:

  • Combine HCK knockdown (using shRNA or CRISPR) with antibody-based detection to confirm specificity

  • Overexpress wild-type or mutant HCK (e.g., gatekeeper mutant HCK T333M) to study resistance mechanisms

What are the best practices for using HCK antibodies in immunohistochemistry?

For optimal immunohistochemistry results with HCK antibodies:

• Tissue preparation:

  • Use formalin-fixed, paraffin-embedded (FFPE) tissue sections (4-6 μm thickness)

  • Include positive control tissues with known HCK expression (e.g., spleen, which shows strong HCK expression)

• Antigen retrieval:

  • Perform heat-induced epitope retrieval (HIER) using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0)

  • Optimize retrieval time (typically 15-20 minutes) for your specific tissue and antibody

• Blocking and antibody incubation:

  • Block endogenous peroxidase activity with 3% H₂O₂

  • Apply protein block to reduce background

  • Use optimized antibody dilution (typically 1:100 to 1:500 for commercial antibodies)

  • Incubate primary antibody overnight at 4°C or 1-2 hours at room temperature

• Detection system:

  • Use a sensitive detection system compatible with your primary antibody species (e.g., HRP-polymer)

  • Develop with DAB and counterstain with hematoxylin

  • Include negative controls (primary antibody omission and isotype control)

• Analysis considerations:

  • Evaluate both intensity and distribution of staining

  • Consider subcellular localization (cytoplasmic, membrane, nuclear)

  • Quantify using appropriate scoring systems (H-score, Allred score) for comparative studies

How can researchers effectively study HCK's role in the tumor microenvironment?

To investigate HCK's role in the tumor microenvironment, implement these advanced methodologies:

• Multiplex immunofluorescence:

  • Co-stain for HCK along with markers of different immune cell populations (CD68+ macrophages, CD11b+ myeloid cells)

  • Use phospho-specific HCK antibodies to identify activated cells within the microenvironment

  • Quantify spatial relationships between HCK-expressing cells and tumor cells

• 3D co-culture systems:

  • Establish co-cultures of tumor cells with macrophages or other immune cells

  • Monitor HCK activation status using phospho-specific antibodies during co-culture

  • Assess podosome formation and ECM degradation, which HCK has been shown to regulate in macrophages

• In vivo models:

  • Use tissue from mouse models (like the HCK^CA mice that develop lung adenocarcinomas as a consequence of lung inflammation)

  • Compare HCK expression and activation in tumor-associated macrophages versus normal tissue macrophages

  • Correlate HCK expression with markers of tumor progression

• Functional readouts:

  • Measure cytokine production (particularly TNFα) in relation to HCK activation status

  • Assess the impact of HCK inhibition on immune cell recruitment and polarization

  • Evaluate changes in extracellular matrix degradation, which HCK-expressing macrophages facilitate

What are the methodological considerations when using HCK antibodies to study resistance mechanisms to targeted therapies?

When investigating HCK-related resistance mechanisms to targeted therapies:

• Target engagement studies:

  • Use biotinylated versions of inhibitors (as done with ibrutinib and CC-292) to pull down HCK and confirm direct binding

  • Perform KiNativ profiling to determine inhibitor engagement in living cells by measuring protection from ATP-biotin probe labeling

• Resistance model development:

  • Generate cell lines expressing inhibitor-resistant HCK mutants (e.g., HCK T333M gatekeeper mutant)

  • Compare antibody-detected phosphorylation patterns between wild-type and mutant HCK-expressing cells

  • Monitor changes in downstream signaling networks

• Combination therapy assessment:

  • Use phospho-specific HCK antibodies to monitor HCK activation status during treatment with multiple inhibitors

  • Track correlation between HCK phosphorylation and cell survival markers

• Patient sample analysis:

  • Compare HCK activation in samples from treatment-naïve versus relapsed/refractory patients

  • Correlate HCK activation with clinical outcomes and treatment response

• Pathway crosstalk investigation:

  • Study HCK's role in facilitating TLR/BCR crosstalk through SYK activation in response to mutated MYD88

  • Use multi-parameter antibody panels to delineate compensatory signaling pathways

How can HCK antibodies be used to understand the role of HCK in normal versus malignant hematopoiesis?

To investigate HCK's differential roles in normal versus malignant hematopoiesis:

• Cell subset analysis:

  • Compare HCK expression and activation across different hematopoietic cell lineages using flow cytometry

  • Create a baseline expression map in healthy donor B cells, memory B cells, and plasma cells

  • Compare to malignant counterparts in diseases like Waldenström macroglobulinemia

• Signaling network analysis:

  • Use phospho-specific HCK antibodies to map activation patterns in response to specific stimuli (e.g., LPS, IFNγ)

  • Compare signaling responses between normal and malignant cells

  • Investigate the relationship between HCK activation and downstream effects on AKT, ERK, and BTK

• Transcriptional regulation studies:

  • Combine HCK protein analysis with transcriptional assessment (e.g., qRT-PCR)

  • Study how HCK transcription is influenced by factors like IL-6 or MYD88 mutations

  • Compare regulatory mechanisms between normal and malignant cells

• Differentiation studies:

  • Monitor HCK expression and activation during normal hematopoietic differentiation

  • Assess changes in cellular localization of HCK during differentiation using immunofluorescence

  • Evaluate the impact of HCK inhibition or knockdown on differentiation potential

What are common challenges when working with HCK antibodies and how can they be overcome?

Common challenges with HCK antibodies and their solutions include:

How can researchers optimize experimental conditions for detecting HCK activation in primary patient samples?

For optimal detection of HCK activation in primary patient samples:

• Sample handling optimization:

  • Process samples immediately after collection to preserve phosphorylation status

  • If immediate processing is not possible, use phosphorylation preservation reagents

  • For bone marrow samples, use gentle RBC lysis buffers to maintain cell integrity

• Phosflow protocol refinement:

  • Optimize fixation time (8-12 minutes at 37°C) with BD Phosflow Fix Buffer I

  • Use gentle permeabilization conditions to maintain epitope accessibility

  • For bone marrow mononuclear cells, include CD19–APC-cy7 antibody for identifying B cells

• Multi-parameter analysis:

  • Include viability dyes to exclude dead cells

  • Use lineage markers to identify specific populations of interest

  • Incorporate markers of activation status (e.g., CD86) to correlate with HCK activation

• Signal amplification strategies:

  • Consider signal amplification systems for samples with low HCK expression

  • Optimize secondary antibody concentration and incubation time

  • Use high-sensitivity detection reagents

• Standardization across samples:

  • Include internal controls (healthy donor samples) in each experimental run

  • Use reference standards for flow cytometry calibration

  • Normalize data to account for inter-sample variability

How might single-cell approaches advance our understanding of HCK biology?

Single-cell approaches offer transformative potential for HCK research:

• Single-cell phospho-profiling:

  • Apply mass cytometry (CyTOF) with HCK antibodies to simultaneously analyze multiple phosphorylation events at single-cell resolution

  • Identify rare subpopulations with unique HCK activation signatures

  • Map signaling heterogeneity within seemingly homogeneous cell populations

• Spatial transcriptomics integration:

  • Combine HCK protein detection with spatial transcriptomics

  • Correlate HCK protein levels with transcriptional networks in individual cells

  • Map HCK-expressing cells within tissue microenvironments

• Live-cell imaging approaches:

  • Develop HCK biosensors for real-time activation monitoring

  • Track HCK translocation and activation dynamics in response to stimuli

  • Correlate temporal activation patterns with functional outcomes

• Single-cell multi-omics:

  • Integrate HCK protein detection with single-cell RNA-seq and ATAC-seq

  • Identify transcriptional and epigenetic regulators of HCK in individual cells

  • Discover novel regulatory mechanisms specific to cell subsets

What are the emerging applications of HCK antibodies in precision medicine approaches?

Emerging precision medicine applications for HCK antibodies include:

• Patient stratification biomarkers:

  • Develop standardized protocols for measuring HCK activation in patient samples

  • Correlate HCK activation profiles with treatment response in clinical trials

  • Identify patient subgroups that might benefit from HCK-targeted therapies

• Resistance mechanism identification:

  • Monitor changes in HCK activation during treatment to detect emerging resistance

  • Identify compensatory signaling pathways activated upon HCK inhibition

  • Develop rational combination therapy approaches based on HCK activation status

• Therapeutic antibody development:

  • Design antibodies that specifically target HCK for therapeutic applications

  • Develop antibody-drug conjugates targeting HCK-expressing cells

  • Create bispecific antibodies linking HCK-expressing cells to immune effectors

• Ex vivo drug sensitivity testing:

  • Use HCK activation status as a readout for ex vivo drug sensitivity testing

  • Develop standardized assays for clinical implementation

  • Correlate ex vivo findings with in vivo treatment responses

• Liquid biopsy applications:

  • Detect HCK in circulating tumor cells or exosomes as biomarkers

  • Monitor treatment response through changes in HCK activation in accessible samples

  • Develop minimally invasive methods for longitudinal patient monitoring