STK4 Antibody, HRP conjugated

What is STK4 and what biological pathways does it participate in?

STK4 (Serine/Threonine Kinase 4, formerly known as MST1) is a stress-activated, pro-apoptotic kinase that serves as a key component of the Hippo signaling pathway. This pathway plays a pivotal role in organ size control and tumor suppression by restricting proliferation and promoting apoptosis. The core of this pathway involves STK3/MST2 and STK4/MST1, which in complex with the regulatory protein SAV1, phosphorylate and activate LATS1/2 in complex with MOB1, which subsequently phosphorylates and inactivates the YAP1 oncoprotein and WWTR1/TAZ .

STK4 also phosphorylates transcription factors in the FOXO family, including FOXO1 and FOXO3, as part of a stress-response pathway. Additionally, STK4 interacts with NORE1A and RASSF1A, which inhibit its kinase activity, and with RAPL, which is essential for lymphocyte polarization and adhesion .

What are the typical applications for STK4-HRP conjugated antibodies?

STK4-HRP conjugated antibodies are versatile tools primarily used in the following applications:

• Western Blotting (WB): For detection of STK4 protein in cell or tissue lysates, with recommended dilutions typically ranging from 1:100-1000 .

• Immunohistochemistry (IHC): For visualization of STK4 protein in tissue sections, with typical dilutions of 1:50-500 .

The HRP conjugation eliminates the need for a secondary antibody, simplifying protocols and potentially reducing background signals. This direct detection approach is particularly valuable when working with limited samples or when trying to minimize cross-reactivity issues .

What are the optimal storage conditions for maintaining STK4-HRP antibody activity?

STK4-HRP conjugated antibodies should be stored at -20°C to maintain optimal activity . Most commercially available antibodies are supplied in a liquid format with stabilizing buffers containing:

• PBS pH 7.4

• 50% glycerol (as a cryoprotectant)

• 0.09% sodium azide (as a preservative)

When handling these antibodies, it's important to note that sodium azide is a hazardous substance that should be handled only by trained staff. For short-term storage during experiments, antibodies can be kept at 4°C, but repeated freeze-thaw cycles should be avoided as they can degrade the antibody and reduce the HRP enzymatic activity.

What is the reactivity profile of commercially available STK4-HRP antibodies?

According to the product information, commercially available STK4-HRP antibodies typically demonstrate reactivity across multiple species:

• Human

• Mouse

• Rat

This cross-reactivity is due to the high conservation of STK4 across mammalian species. When selecting an antibody for your research, it's important to verify that the specific epitope recognized by the antibody is conserved in your species of interest. For example, the anti-STK4 antibody described in search result targets amino acids 320-420 of human STK4, which corresponds to a region that shows high homology across species .

How can I validate the specificity of STK4-HRP antibodies for my experiments?

Validating antibody specificity is crucial for reliable experimental results. Several approaches can be used:

• Western blot analysis: The anti-STK4 antibody should detect a protein band at approximately 55.6 kDa in human samples, as demonstrated in human cervical cancer cell line (HeLa) lysates .

• Positive controls: Include samples known to express STK4, such as HeLa cells.

• Negative controls: Either use samples from STK4-deficient patients/models or perform pre-absorption tests with the target peptide (as demonstrated in the substance P antibody study where preabsorption with substance P obliterated the reaction) .

• Comparative analysis: Compare staining patterns with other validated STK4 antibodies targeting different epitopes.

How can STK4-HRP antibodies be optimized for studying the Hippo signaling pathway?

Studying the Hippo signaling pathway using STK4-HRP antibodies requires careful optimization of experimental conditions:

• Phosphorylation-specific detection: Since STK4's function involves phosphorylation cascades, researchers should consider using phospho-specific antibodies alongside total STK4 antibodies to monitor activation states.

• Multi-protein analysis: For comprehensive pathway analysis, use STK4-HRP antibodies in conjunction with antibodies against other pathway components (SAV1, LATS1/2, MOB1, YAP1, and WWTR1/TAZ) .

• Co-immunoprecipitation studies: When investigating protein interactions in the Hippo pathway, optimize buffer conditions to preserve physiologically relevant interactions.

• In situ proximity ligation assays: These can be performed using STK4-HRP antibodies in combination with antibodies against interaction partners to visualize protein complexes within cells.

• Subcellular fractionation: STK4 translocates to the nucleus following caspase-cleavage, so separate analysis of cytoplasmic and nuclear fractions may provide additional insights into pathway activation .

What are the optimal conditions for immunohistochemical staining using STK4-HRP antibodies?

Based on the available research data, optimal immunohistochemical staining with HRP-conjugated antibodies requires careful optimization:

• Fixation: Formalin-fixed paraffin-embedded (FFPE) tissue sections are compatible with STK4-HRP antibody staining .

• Antigen retrieval: This step is critical for FFPE tissues to expose epitopes masked during fixation.

• Dilution optimization: Start with the manufacturer's recommended dilution (typically 1:50 for IHC) and adjust based on signal-to-noise ratio.

• Incubation conditions: Evidence from similar HRP-conjugated antibody studies suggests that prolonged incubations at lower temperatures (12°C) in the presence of 0.1% Triton X-100 can yield better immunohistochemical results .

• Detection system: Use HRP-DAB (3,3'-diaminobenzidine) detection system for visualization of antibody binding .

• Counterstaining: Hematoxylin counterstaining provides contrast to visualize tissue morphology alongside the DAB signal .

How do STK4-HRP antibodies compare in detecting STK4 in different cell populations relevant to immunodeficiency studies?

STK4 deficiency affects multiple immune cell populations, making antibody performance across different cell types an important consideration:

• T cells: STK4-deficient patients show reduced naive T cells (CD45RA+CD45RO-) and central memory T cells (CD62L+CCR7+), while effector memory T cells (CD62L-CCR7-) are less affected . STK4-HRP antibodies can be used to correlate protein expression with these phenotypic changes.

• B cells: Patients exhibit decreased CD19+ B cells, with alterations in transitional B cells (CD38++IgM high), marginal zone B cells (IgD+IgM+CD27+), and switched memory B cells (IgD-IgM-CD27+) . Western blot analysis of sorted B cell populations can reveal differential STK4 expression patterns.

• Neutrophils: Despite previous reports suggesting lack of STK4 expression in neutrophils, Western blot analysis of isolated neutrophil granulocytes from STK4-deficient patients and controls confirms STK4 expression in these cells . This highlights the importance of antibody validation in specific cell populations.

• Challenges in detection: STK4-deficient cells exhibit enhanced susceptibility to apoptosis, which can complicate in vitro studies. Researchers have noted difficulties in maintaining STK4-deficient T cells in culture due to premature cell death .

What experimental controls are essential when using STK4-HRP antibodies in apoptosis studies?

When investigating STK4's role in apoptosis, several critical controls should be included:

• Positive controls for apoptosis: Include samples treated with known apoptosis inducers (e.g., staurosporine).

• Viability controls: Monitor cell death using complementary approaches such as Annexin V/PI staining, TUNEL assay, or caspase activation assays.

• Mitochondrial membrane potential: Since STK4-deficient lymphocytes and neutrophils exhibit enhanced loss of mitochondrial membrane potential, include assessments of this parameter using appropriate dyes .

• Genetic controls: When available, include samples from STK4-deficient models or patients as negative controls for antibody specificity .

• Functional rescue experiments: Although challenging due to the increased susceptibility to apoptosis in STK4-deficient cells, attempts to reconstitute STK4 expression can provide valuable controls for specificity .

• Time-course experiments: Apoptosis is a dynamic process, so analyzing multiple time points is essential for capturing the relevant cellular events.

What are the technical considerations when using STK4-HRP antibodies in multiplex Western blot analyses?

Multiplex Western blot analyses with STK4-HRP antibodies require attention to several technical factors:

• Molecular weight considerations: STK4 has a molecular weight of approximately 55.6 kDa . When designing multiplex experiments, ensure that other proteins of interest have sufficiently different molecular weights to avoid signal overlap.

• Dilution optimization: The recommended dilution for Western blotting is 1:1000 , but this should be optimized when used in combination with other antibodies.

• Stripping and reprobing limitations: HRP conjugation can complicate stripping and reprobing procedures. Consider using separate blots or fluorescent-based multiplex systems if multiple proteins with similar molecular weights need to be detected.

• Development time: When detecting multiple proteins, optimize the development time to ensure adequate signal for STK4 without oversaturating signals from more abundant proteins.

• Loading controls: Include appropriate loading controls (e.g., β-actin, GAPDH) to normalize STK4 expression levels, but ensure these do not overlap with the STK4 signal.

A typical Western blot protocol would involve loading 10 μg of cell lysate per lane, using a 1:1000 dilution of the STK4-HRP antibody, and developing using ECL (enhanced chemiluminescence) detection .