MKL2 Antibody, HRP conjugated

What is an HRP-conjugated antibody and what advantages does it offer in research applications?

HRP (Horseradish Peroxidase) conjugated antibodies are specialized immunological tools where the enzyme HRP is chemically linked to an antibody of interest. This conjugation enables direct detection of target proteins without requiring secondary antibodies, thus streamlining experimental workflows and potentially reducing background signal. When an appropriate substrate is added, HRP catalyzes a reaction that produces a detectable signal (colorimetric, chemiluminescent, or fluorescent), allowing visualization of the target protein.

The primary advantages include simplified protocols, reduced incubation times, minimized cross-reactivity issues, and enhanced signal amplification capabilities. For research applications involving MKLP2 and related proteins, HRP-conjugated antibodies provide efficient detection in various experimental contexts including Western blotting, ELISA, and immunohistochemistry .

What are the main applications of MKLP2 antibody and how does it compare to other HRP-conjugated antibodies?

MKLP2 antibody (HRP conjugated) is primarily used in the following applications:

• Western Blotting (WB) at dilutions of 1:300-5000

• Enzyme-Linked Immunosorbent Assay (ELISA) at dilutions of 1:500-1000

• Immunohistochemistry on paraffin-embedded tissues (IHC-P) at dilutions of 1:200-400

• Immunohistochemistry on frozen tissues (IHC-F) at dilutions of 1:100-500

When compared to other HRP-conjugated antibodies such as MAPKAPK2 (Thr222) and DYKDDDDK Tag antibodies, MKLP2 antibody shares similar application versatility but targets a distinct protein involved in mitotic processes and Golgi transport rather than stress response pathways or epitope tags .

What species reactivity can be expected when using MKLP2 antibody?

Based on manufacturer specifications, MKLP2 Polyclonal Antibody (HRP Conjugated) demonstrates confirmed reactivity with human and rat samples. Additionally, it is predicted to cross-react with mouse and rabbit samples, though this requires experimental validation in each specific research context .

This reactivity profile is distinct from other HRP-conjugated antibodies in the MAPK pathway, such as MAPKAPK2 antibody which reacts with mouse, rat, and monkey samples with predicted reactivity to human, dog, cow, and rabbit samples .

How does phosphorylation affect the function of proteins in signaling pathways, and how can phospho-specific HRP-conjugated antibodies enhance this research?

Phosphorylation serves as a critical molecular switch in signaling cascades like the MAPK pathway. For example, MAPKAPK2 is activated through phosphorylation at Thr222 by p38-alpha/MAPK14, which then enables MAPKAPK2 to phosphorylate downstream substrates including HSP27/HSPB1, leading to dissociation of heat-shock protein oligomers and altered chaperone activities .

Phospho-specific HRP-conjugated antibodies, such as MAPKAPK2 (Thr222), provide researchers with the ability to:

• Directly detect the activated (phosphorylated) form of the protein

• Quantify activation dynamics in response to cellular stressors

• Track signal propagation through cascades

• Evaluate the efficacy of kinase inhibitors or other pathway modulators

This approach offers significant advantages over total protein detection when studying rapid, reversible signaling events and provides temporal resolution of pathway activation that might be missed with conventional antibodies .

What are the critical considerations for validating specificity of HRP-conjugated antibodies targeting components of complex signaling networks?

For HRP-conjugated antibodies targeting signaling proteins, comprehensive validation should include:

• Genetic validation: Using knockout/knockdown models to confirm specificity, as demonstrated with MEK2 antibody where "signal was lost in MAP2K2 (MEK2) knockout cells"

• Epitope mapping: Understanding the precise region recognized by the antibody and potential cross-reactivity with homologous proteins (particularly important for antibodies like MKLP2 where the immunogen covers a specific region (353-450/890) of the target protein)

• Signal quantification: Establishing clear thresholds for positive detection versus background signal

• Orthogonal validation: Confirming results using alternative methodologies or antibodies targeting different epitopes of the same protein

• Isoform specificity: Verifying the antibody's ability to distinguish between closely related isoforms or family members (particularly important in complex pathways like MAPK signaling)

How can researchers troubleshoot background signal issues and optimize signal-to-noise ratio when using HRP-conjugated antibodies?

Optimizing signal-to-noise ratio requires systematic troubleshooting:

• Antibody titration: Test multiple dilutions around the manufacturer's recommendation (e.g., for MKLP2 antibody, WB: 1:300-5000, ELISA: 1:500-1000, IHC-P: 1:200-400)

• Block optimization: Test different blocking agents (BSA, non-fat milk, commercial blockers) and concentrations to minimize non-specific binding

• Wash optimization: Increase wash duration/frequency or adjust buffer composition (salt concentration, detergent type/concentration)

• Substrate selection: Match HRP substrate sensitivity to target abundance (using high-sensitivity substrates only when necessary)

• Endogenous peroxidase quenching: For tissue samples, include an endogenous peroxidase quenching step (typically using hydrogen peroxide)

• Storage buffer considerations: Check if components in the antibody storage buffer (such as the 50% glycerol, 1% BSA, and 0.03% Proclin300 used for MKLP2 antibody) could contribute to background in specific applications

What is the recommended protocol for Western blotting using HRP-conjugated antibodies targeting components of the MAPK pathway?

The optimal Western blotting protocol for HRP-conjugated antibodies varies slightly by target, but generally includes:

• Sample preparation: Lyse cells in buffer containing appropriate protease and phosphatase inhibitors (critical for phospho-epitopes)

• Protein separation: Separate 20-50μg protein by SDS-PAGE

• Transfer: Transfer proteins to PVDF or nitrocellulose membrane

• Blocking: Block membrane in 5% non-fat milk or BSA in TBST (target-dependent)

• Primary antibody incubation: Dilute antibody according to manufacturer specifications:

  • MKLP2 antibody: 1:300-5000 for WB

  • DYKDDDDK Tag antibody: 1:1000 for WB

  • MEK2 antibody: Specific dilution recommendations provided by manufacturer

• Washing: Wash 3-5 times with TBST

• Detection: Apply appropriate HRP substrate (chemiluminescent, fluorescent, or colorimetric)

• Documentation: Image membrane using appropriate detection system

• Quantification: Normalize to appropriate loading control

What controls should be included when using HRP-conjugated antibodies for quantitative analysis?

Rigorous experimental design requires comprehensive controls:

• Negative controls:

  • No primary antibody control (to assess secondary antibody specificity and background)

  • Isotype control (same species and isotype but irrelevant specificity)

  • Knockout/knockdown samples when available (as demonstrated for MEK2 antibody)

• Positive controls:

  • Samples known to express the target protein

  • Recombinant protein standards or overexpression systems

• Loading/normalization controls:

  • Housekeeping proteins (GAPDH, β-actin, α-tubulin)

  • Total protein normalization for phospho-specific antibodies

• Technical controls:

  • Serial dilution of samples to verify detection linearity

  • Replicate samples to assess reproducibility

  • Batch controls across multiple experiments

How should researchers properly store and handle HRP-conjugated antibodies to maintain optimal activity?

To preserve antibody function and extend shelf-life:

• Storage temperature: Maintain at -20°C as recommended for MKLP2, MAPKAPK2, and other HRP-conjugated antibodies

• Aliquoting: Divide into small, single-use aliquots to avoid repeated freeze-thaw cycles which can significantly degrade HRP enzyme activity and antibody binding

• Buffer conditions: Store in manufacturer-provided buffer containing stabilizers (typical buffers contain 0.01M TBS (pH 7.4), 1% BSA, 0.03% Proclin300, and 50% glycerol)

• Light protection: Shield from light during storage and handling to prevent photobleaching

• Working dilution handling: Prepare fresh working dilutions for each experiment rather than storing diluted antibody

• Contamination prevention: Use sterile technique when handling to prevent microbial contamination

Comparative Analysis of HRP-Conjugated Antibodies for MAPK Pathway Research

Detailed Specifications for MKLP2 Antibody

Functional Characteristics of MKLP2/KIF20A

MKLP2 (also known as KIF20A) functions as a mitotic kinesin required for chromosome passenger complex (CPC)-mediated cytokinesis. Following phosphorylation by PLK1, it is involved in the recruitment of PLK1 to the central spindle. The protein interacts with GTP-bound forms of RAB6A and RAB6B and may act as a motor required for the retrograde RAB6-regulated transport of Golgi membranes and associated vesicles along microtubules. MKLP2 demonstrates microtubule plus end-directed motility .

How can researchers effectively use HRP-conjugated antibodies to study protein phosphorylation dynamics in response to cellular stress?

To study phosphorylation dynamics with HRP-conjugated antibodies:

• Time-course experiments: Treat cells with stressors and collect samples at multiple time points to track phosphorylation kinetics

• Stimulus titration: Vary stimulus concentration to establish dose-response relationships

• Inhibitor studies: Use pathway-specific inhibitors to confirm signaling mechanisms

• Parallel detection strategies:

  • Use phospho-specific antibodies (like MAPKAPK2 (Thr222)) alongside total protein antibodies

  • Compare multiple phosphorylation sites on the same protein

  • Examine multiple proteins within the same pathway

• Normalization approach:

  • Normalize phospho-signal to total protein rather than housekeeping genes

  • Consider using total protein stains for more accurate normalization

• Quantification methods:

  • Use linear range of detection for accurate quantification

  • Apply appropriate statistical analyses for time-course data

What methodological approaches can resolve contradictory data when using different antibodies targeting the same signaling pathway?

When faced with contradictory results:

• Epitope mapping: Compare the specific epitopes recognized by different antibodies and consider how protein conformation, post-translational modifications, or protein-protein interactions might affect epitope accessibility

• Validation rigor: Assess the validation evidence for each antibody, prioritizing those with genetic validation (knockout/knockdown controls) and extensive characterization

• Application optimization: Evaluate whether each antibody has been optimized for the specific application used (WB, IHC, etc.)

• Technical replication: Increase technical and biological replicates to distinguish true biological differences from technical artifacts

• Orthogonal approaches: Implement non-antibody-based methods (mass spectrometry, CRISPR screens, etc.) to resolve discrepancies

• Contextual factors: Consider cell type differences, culture conditions, and experimental timing that might explain apparently contradictory results