MPP1 Antibody, HRP conjugated

What is the difference between polyclonal and monoclonal anti-MPP1 antibodies, and when should each be used?

The primary difference between polyclonal and monoclonal anti-MPP1 antibodies lies in their specificity and production methods:

Polyclonal anti-MPP1 antibodies:

• Produced by multiple B cell lineages in an immunized animal (typically rabbit)

• Recognize multiple epitopes on the MPP1 protein

• Offer higher sensitivity due to binding to multiple epitopes

• Example: The rabbit polyclonal MPP1 antibody (ab96255) is suitable for Western blot (WB) and immunohistochemistry on paraffin-embedded sections (IHC-P)

Monoclonal anti-MPP1 antibodies:

• Produced by a single B cell clone

• Recognize a single epitope on the MPP1 protein

• Provide higher specificity and consistency between batches

• Example: The rabbit recombinant monoclonal MPP1 antibody [EPR5865] (ab108528) demonstrates high specificity for MPP1 in wild-type HEK293T cells with loss of signal in MPP1 knockout cells

Researchers should choose polyclonal antibodies when prioritizing signal strength and detection of proteins in native conditions or denatured states. Monoclonal antibodies are preferable for applications requiring high specificity, reproducibility between experiments, and when differentiating between closely related proteins . For novel investigations of MPP1, using both types in parallel can provide complementary information.

How does HRP conjugation affect the functionality of anti-MPP1 antibodies?

HRP (Horseradish Peroxidase) conjugation provides anti-MPP1 antibodies with enzymatic activity that enables direct detection through colorimetric, chemiluminescent, or fluorescent substrates without requiring secondary antibodies. The conjugation process may affect antibody functionality in several ways:

• Detection sensitivity: HRP conjugation enhances detection sensitivity, particularly in applications like ELISA and Western blotting, as the enzymatic amplification of signal allows for detection of lower quantities of MPP1 .

• Epitope accessibility: The conjugation process may potentially affect the antibody's antigen binding site depending on the conjugation chemistry used. Commercial anti-MPP1-HRP conjugates are optimized to minimize this impact .

• Stability considerations: HRP-conjugated antibodies typically have shorter shelf lives than unconjugated antibodies. Proper storage and the addition of stabilizers (such as LifeXtend™ HRP Conjugate Stabilizer/Diluent) are crucial for maintaining activity over time .

• Dilution recommendations: HRP-conjugated anti-MPP1 antibodies require specific dilution parameters for optimal performance: 1:1000 for ELISA, 1:100-500 for Western blot, 1:50-100 for immunochemistry, and 1:10-50 for flow cytometry .

The benefits of direct detection must be weighed against potential limitations in signal amplification that might be achieved through secondary antibody approaches when working with samples containing low abundance MPP1.

What are the optimal conditions for using HRP-conjugated anti-MPP1 antibodies in Western blotting?

Optimizing Western blot protocols for HRP-conjugated anti-MPP1 antibodies requires attention to several key parameters:

Sample preparation and loading:

• Use fresh cell lysates when possible; HeLa cells are a validated source for MPP1 detection

• Load approximately 30 μg of total protein per lane for standard detection

• Use 10% SDS-PAGE gels for optimal separation of MPP1 (expected molecular weight: 52-55 kDa)

Blocking and antibody incubation:

• Block membranes with 5% non-fat dry milk or BSA in TBST for 1 hour at room temperature

• Dilute HRP-conjugated anti-MPP1 antibody to 1:100-500 in blocking buffer

• Incubate membrane with diluted antibody overnight at 4°C for optimal binding

• Wash extensively (4-5 times for 5 minutes each) with TBST

Detection and visualization:

• Use fresh ECL substrate to minimize background

• Optimize exposure time based on signal intensity (typically 30 seconds to 5 minutes)

• Consider using LifeXtend™ HRP Conjugate Stabilizer/Diluent if preparing working dilutions for extended use

For verification of specificity, include appropriate controls such as MPP1 knockout cell lysates, which should show complete absence of the target band as demonstrated with the ab108528 antibody in HEK293T cells .

How can HRP-conjugated anti-MPP1 antibodies be used to study MPP1's interaction with ABCC4 in leukemia research?

HRP-conjugated anti-MPP1 antibodies are valuable tools for investigating the clinically significant interaction between MPP1 and ABCC4 in leukemia research. Based on established methodologies, the following approaches can be employed:

Co-immunoprecipitation studies:

• Use HRP-conjugated anti-MPP1 antibodies to detect MPP1 after immunoprecipitation with anti-ABCC4 antibodies (or vice versa)

• This approach has successfully demonstrated that endogenous ABCC4 and MPP1 physically associate in myeloid leukemia cells, as shown in MO7e cells (a pediatric myeloid leukemia cell line)

• Include appropriate controls, such as non-specific antibodies, which should not co-immunoprecipitate either protein

Functional studies of MPP1-ABCC4 interaction:

• Use HRP-conjugated anti-MPP1 antibodies in Western blotting to assess MPP1 expression in relation to ABCC4 levels in patient samples

• Employ membrane fractionation followed by Western blotting to examine how MPP1 affects ABCC4 membrane localization

• This approach helped establish that MPP1 increases ABCC4 membrane retention through a protein complex formation dependent on ABCC4's PDZ-binding motif

Therapeutic intervention studies:

• Use HRP-conjugated anti-MPP1 antibodies to monitor changes in MPP1-ABCC4 interaction after treatment with potential disruptors of this protein complex

• This method was instrumental in identifying Antimycin A as a small molecule that disrupts the ABCC4-MPP1 protein complex and reverses drug resistance in AML cell lines and primary patient AML cells

These approaches collectively support investigation of the MPP1-ABCC4 axis as a potential therapeutic target in AML, where long-term prognosis remains poor.

What are the recommended protocols for using HRP-conjugated anti-MPP1 antibodies in ELISA assays?

When designing ELISA protocols with HRP-conjugated anti-MPP1 antibodies, the following methodological approach is recommended for optimal results:

Direct ELISA protocol:

• Coating: Adsorb purified MPP1 protein or cellular extract containing MPP1 onto high-binding ELISA plates in carbonate-bicarbonate buffer (pH 9.6) overnight at 4°C

• Blocking: Block non-specific binding sites with 2-5% BSA or non-fat dry milk in PBS-T (PBS with 0.05% Tween-20) for 1-2 hours at room temperature

• Antibody incubation: Dilute HRP-conjugated anti-MPP1 antibody to 1:1000 in blocking buffer and incubate for 1-2 hours at room temperature

• Washing: Perform thorough washing (4-5 times) with PBS-T between each step to minimize background

• Detection: Add TMB (3,3',5,5'-tetramethylbenzidine) substrate and incubate for 10-30 minutes, then stop the reaction with 2N H₂SO₄ or 1N HCl

• Measurement: Read absorbance at 450 nm with reference wavelength at 620 nm

Sandwich ELISA considerations:

For a sandwich ELISA, use an unconjugated capture anti-MPP1 antibody that recognizes a different epitope than the HRP-conjugated anti-MPP1 detection antibody. This approach is particularly useful for quantifying MPP1 in complex biological samples.

Important optimization parameters:

• Include a standard curve using recombinant MPP1 protein at concentrations ranging from 0-1000 ng/mL

• Run all samples and standards in duplicate or triplicate

• Include appropriate negative controls (buffer only) and positive controls (known MPP1-positive samples)

• If working with dilute samples, consider concentrating the antibody using an Antibody Concentration and Clean Up Kit, as concentrations below 0.5 mg/mL may reduce conjugation efficiency

This protocol can be adapted for investigating MPP1 levels in patient samples, such as those from AML patients, where MPP1 expression correlates with clinical outcomes .

What are common troubleshooting approaches for weak or absent signals when using HRP-conjugated anti-MPP1 antibodies?

When encountering weak or absent signals with HRP-conjugated anti-MPP1 antibodies, systematic troubleshooting should address several potential issues:

Antibody-related factors:

• Degradation or inactivation: HRP conjugates have limited stability. Verify storage conditions (typically 4°C, avoid freeze-thaw cycles) and expiration date. Consider testing antibody activity using a dot blot with known positive control .

• Improper dilution: Ensure proper antibody dilution; too dilute solutions may result in weak signals. For anti-MPP1-HRP antibodies, verified dilutions are: ELISA (1:1000), Western blot (1:100-500), immunochemistry (1:50-100), and flow cytometry (1:10-50) .

• Buffer incompatibility: Certain buffer components (BSA, Tris, azide, glycine) can interfere with HRP activity. If necessary, perform buffer exchange using an Antibody Concentration and Clean Up Kit .

Sample-related factors:

• Low target expression: MPP1 expression varies across cell types. HeLa cells are validated for MPP1 detection . For unknown samples, include positive controls with confirmed MPP1 expression.

• Protein degradation: Use fresh samples with protease inhibitors and keep samples cold during preparation. For stored samples, verify protein integrity on total protein stains.

• Epitope masking: Ensure sample preparation conditions (lysis buffers, fixation methods) preserve the epitope recognized by the antibody.

Technique-specific factors:

• Western blotting: Optimize transfer conditions, as MPP1 (52-55 kDa) may require specific transfer parameters. Verify transfer efficiency using reversible protein stains.

• Substrate limitations: Prepare fresh ECL substrate according to manufacturer instructions. Expired or improperly stored substrates can significantly reduce signal.

Systematic approach to resolution:

Begin with control experiments using known positive samples at various antibody dilutions. If problems persist, systematically modify one variable at a time (blocking reagent, incubation time, washing stringency) while keeping others constant to identify the source of the problem.

How can researchers optimize HRP conjugation to anti-MPP1 antibodies in-house?

For laboratories performing in-house conjugation of HRP to anti-MPP1 antibodies, the following optimization protocol is recommended to achieve optimal conjugation efficiency:

Prerequisite conditions:

• Antibody concentration: Start with anti-MPP1 antibody at a concentration greater than 0.5 mg/mL. Lower concentrations significantly reduce conjugation efficiency .

• Buffer compatibility: Ensure the antibody is in a buffer free from incompatible additives. Common problematic additives include:

  • BSA (competes for conjugation sites)

  • Tris (contains primary amines that interfere with cross-linking)

  • Sodium azide (inhibits HRP activity)

  • Glycine (competes with conjugation chemistry)

• Antibody purity: Higher purity antibodies yield better conjugates. Consider purification if the starting material contains stabilizing proteins.

Optimization protocol:

• Buffer exchange: If necessary, perform buffer exchange using an Antibody Concentration and Clean Up Kit to remove interfering additives while concentrating the antibody .

• Conjugation ratio optimization: Test different molar ratios of HRP to antibody (typically 2:1, 4:1, and 6:1) to determine the optimal ratio that maintains antibody specificity while providing sufficient enzymatic activity.

• Reaction conditions:

  • Maintain pH between 7.2-7.4 for optimal conjugation

  • Perform reaction at room temperature (20-25°C)

  • Allow sufficient reaction time (typically 1-3 hours) as per conjugation kit instructions

• Purification of conjugates: Remove unreacted HRP using appropriate size-exclusion methods calibrated for the ~52 kDa MPP1 antibody plus ~44 kDa HRP.

• Stabilization: Add stabilizing reagents to the final conjugate such as LifeXtend™ HRP Conjugate Stabilizer/Diluent for long-term storage .

Quality control assessment:

• Activity testing: Verify both antibody binding and HRP activity using known MPP1-positive samples (e.g., HeLa cell lysates) .

• Specificity verification: Confirm specificity using MPP1 knockout samples as negative controls, similar to the validation performed with commercial antibodies .

• Stability testing: Assess conjugate stability by testing activity after storage under recommended conditions at various time intervals.

This systematic approach will help researchers optimize in-house HRP conjugation to anti-MPP1 antibodies while minimizing common problems that lead to poor conjugate performance.

What are the critical buffer compatibility considerations when working with HRP-conjugated anti-MPP1 antibodies?

Buffer composition significantly impacts the performance of HRP-conjugated anti-MPP1 antibodies. Understanding these compatibility issues is essential for maintaining antibody functionality and enzymatic activity:

Problematic buffer components and their effects:

Recommended buffer systems:

• Storage buffer: Phosphate-buffered saline (PBS, pH 7.2-7.4) containing:

  • 50% glycerol for stabilization

  • Alternative preservatives like ProClin™ or thimerosal instead of sodium azide

  • Consider commercial stabilizers like LifeXtend™ HRP Conjugate Stabilizer/Diluent for long-term storage

• Working buffer for Western blotting: Tris-buffered saline with 0.05-0.1% Tween-20 (TBST) at pH 7.4

• Working buffer for ELISA: Phosphate-buffered saline with 0.05% Tween-20 (PBST) at pH 7.4

Special considerations for MPP1 applications:

When investigating MPP1's interaction with ABCC4 in membrane fractions, specialized buffers may be required that contain detergents for membrane protein solubilization. In such cases, carefully test detergent compatibility with the HRP conjugate and consider using higher antibody concentrations to overcome potential interference .

Proper buffer selection will maximize both the sensitivity and specificity of HRP-conjugated anti-MPP1 antibodies across various experimental applications.

How can HRP-conjugated anti-MPP1 antibodies be applied to investigate MPP1's role in drug resistance mechanisms in cancer?

HRP-conjugated anti-MPP1 antibodies provide valuable tools for investigating MPP1's critical role in drug resistance, particularly in the context of acute myeloid leukemia (AML). The following methodological approaches leverage these antibodies to elucidate resistance mechanisms:

Membrane localization studies:

• Subcellular fractionation analysis: Use HRP-conjugated anti-MPP1 antibodies to detect MPP1 in membrane vs. cytosolic fractions after treatment with chemotherapeutic agents. This approach helped establish that MPP1 increases ABCC4 membrane localization and retention, enhancing drug resistance .

• Confocal microscopy: While immunofluorescence typically uses fluorophore-conjugated antibodies, HRP-conjugated antibodies with tyramide signal amplification can provide enhanced sensitivity for detecting MPP1-ABCC4 colocalization in resistant vs. sensitive cells.

Protein-protein interaction disruption studies:

• Co-immunoprecipitation with drug treatments: Use HRP-conjugated anti-MPP1 antibodies in Western blot analysis following immunoprecipitation to assess how potential therapeutic compounds affect the MPP1-ABCC4 interaction. This approach was instrumental in evaluating how Antimycin A disrupts the ABCC4-MPP1 protein complex .

• PDZ domain mutation analysis: Investigate how mutations in MPP1's PDZ domain affect its interaction with ABCC4 by comparing wild-type vs. mutant proteins using HRP-conjugated anti-MPP1 antibodies. Research has shown that the PDZ-binding motif of ABCC4 is required for forming the protein complex with MPP1 .

Clinical correlation studies:

These methodologies collectively provide a comprehensive approach to understanding MPP1's role in drug resistance mechanisms, potentially leading to novel therapeutic strategies targeting the MPP1-ABCC4 axis in AML and possibly other cancers.

What are the considerations for using HRP-conjugated anti-MPP1 antibodies in multiplex immunoassays to study neutrophil polarization pathways?

Multiplex immunoassays combining HRP-conjugated anti-MPP1 antibodies with other detection systems present advanced opportunities for studying neutrophil polarization pathways. Careful methodological considerations are required to overcome technical challenges:

Assay design considerations:

• Enzyme/fluorophore selection: When combining HRP-conjugated anti-MPP1 antibodies with other detection systems, choose complementary reporters with non-overlapping detection parameters:

  • Pair HRP (detected via chemiluminescence) with fluorophores having emission spectra distinct from HRP substrates

  • Consider sequential detection protocols to prevent signal interference

• Epitope accessibility: MPP1 functions as an essential regulator of neutrophil polarity, regulating neutrophil polarization through AKT1 phosphorylation . Design multiplex panels to include antibodies against different components of this pathway (PIK3CG, AKT1, phospho-AKT1) that target distinct, non-competing epitopes.

• Signal separation strategies:

  • Temporal separation: Detect HRP signals first before moving to fluorescent detection

  • Spatial separation: Use tyramide signal amplification with HRP to deposit distinct fluorophores at different locations

  • Spectral separation: Use narrow bandwidth filters to distinguish between detection systems

Protocol optimization for neutrophil studies:

• Sample preparation: Neutrophils require specialized handling due to their short half-life and propensity for activation:

  • Use rapid isolation protocols with minimal manipulation

  • Consider fixation methods that preserve MPP1 epitopes while maintaining cellular architecture

  • For polarization studies, capture neutrophils in various activation states

• Controls and normalization:

  • Include phosphatase inhibitors when studying phosphorylation-dependent pathways

  • Use housekeeping proteins as internal controls for normalization

  • Include single-stained controls to establish bleed-through parameters

• Quantitative analysis:

  • Employ digital image analysis algorithms for colocalization studies

  • Use ratiometric approaches to measure relative distribution of MPP1 during polarization

  • Develop stimulation protocols that induce gradual polarization to capture dynamic changes

This advanced multiplex approach enables researchers to simultaneously monitor MPP1 localization and its interaction with signaling partners during neutrophil polarization, providing insights into the mechanism independent of PIK3CG activity as described in the literature .

How can researchers validate the specificity of HRP-conjugated anti-MPP1 antibodies in complex biological samples?

Validating the specificity of HRP-conjugated anti-MPP1 antibodies in complex biological samples requires a multi-faceted approach to ensure accurate and reliable research outcomes:

Genetic validation approaches:

• Knockout/knockdown controls: The gold standard for antibody validation involves comparing signals between wild-type and MPP1-deficient samples. Commercial antibodies like ab108528 have been validated using MPP1 knockout HEK-293T cell lines, showing complete loss of signal in knockout samples . Researchers should:

  • Generate MPP1 knockdown/knockout models using CRISPR-Cas9 or siRNA approaches

  • Include these as negative controls in experiments

  • Verify knockdown/knockout efficiency using PCR or other genetic methods

• Overexpression systems: Complement knockout studies with overexpression models:

  • Create cell lines with controlled MPP1 overexpression

  • Verify signal increase proportional to expression level

  • Include tagged versions (His, FLAG, etc.) for dual detection strategies

Biochemical validation approaches:

• Peptide competition assays: Pre-incubate HRP-conjugated anti-MPP1 antibodies with increasing concentrations of purified MPP1 protein or immunogenic peptide before application to samples. Specific antibodies will show dose-dependent signal reduction.

• Cross-reactivity assessment: Test antibodies on samples from multiple species with varying degrees of MPP1 homology to establish specificity boundaries. Commercial anti-MPP1 antibodies are typically validated for human samples .

• Multiple antibody verification: Compare results using different anti-MPP1 antibodies targeting distinct epitopes:

  • Use both polyclonal (e.g., ab96255) and monoclonal (e.g., ab108528) antibodies

  • Confirm that both antibodies produce similar patterns in the same samples

Technical validation approaches:

• Molecular weight verification: Confirm that the detected band in Western blotting matches the expected molecular weight of MPP1 (52-55 kDa) .

• Tissue/cell type expression pattern: Compare detection patterns with known MPP1 expression profiles from transcriptomic data. MPP1 has been extensively studied in hematopoietic cells, particularly in relation to AML .

• Immunodepletion studies: Sequentially deplete samples of MPP1 using unconjugated antibodies, then probe with HRP-conjugated anti-MPP1 antibodies to confirm signal reduction.

This comprehensive validation strategy ensures that findings based on HRP-conjugated anti-MPP1 antibodies accurately reflect MPP1 biology rather than non-specific interactions or artifacts.

How might HRP-conjugated anti-MPP1 antibodies facilitate investigation of MPP1 as a therapeutic target in AML?

HRP-conjugated anti-MPP1 antibodies can significantly advance research exploring MPP1 as a therapeutic target in acute myeloid leukemia (AML) through several methodological approaches:

Target validation studies:

Therapeutic intervention assessment:

• Protein-protein interaction disruption screening: HRP-conjugated anti-MPP1 antibodies are valuable tools for high-throughput screening of compounds that disrupt the MPP1-ABCC4 interaction. This approach led to the identification of Antimycin A as a small molecule that disrupts this protein complex and reverses drug resistance in AML cell lines and primary patient AML cells .

• Mechanistic studies of candidate therapeutics: The antibodies enable detailed investigation of how candidate compounds affect MPP1's subcellular localization and interaction network. Research has shown that the highly conserved PDZ-binding motif of ABCC4 is required for forming a protein complex with MPP1, which increases ABCC4 membrane localization and retention, enhancing drug resistance .

Combination therapy development:

• Synergy assessment: HRP-conjugated anti-MPP1 antibodies can be used to monitor changes in MPP1-ABCC4 interaction when combining MPP1-targeting agents with standard chemotherapeutics, potentially identifying synergistic combinations.

• Resistance mechanism elucidation: These antibodies help track alterations in MPP1 expression or localization during treatment, providing insights into resistance mechanisms that might emerge during therapy.

This multifaceted approach using HRP-conjugated anti-MPP1 antibodies supports the development of novel therapies targeting the MPP1-ABCC4 axis to improve treatment outcomes in AML, a disease where long-term prognosis remains poor .

What are the methodological approaches for investigating post-translational modifications of MPP1 using HRP-conjugated antibodies?

Investigating post-translational modifications (PTMs) of MPP1 requires sophisticated methodological approaches that leverage the sensitivity and specificity of HRP-conjugated antibodies:

Palmitoylation analysis:

MPP1 (Membrane Protein, Palmitoylated 1) is known to undergo palmitoylation, a critical modification for its membrane association. To study this modification:

• Acyl-biotin exchange (ABE) assay combined with immunoblotting:

  • Treat samples with hydroxylamine to cleave palmitoyl groups

  • Label newly exposed thiols with biotin-BMCC

  • Capture biotinylated proteins with streptavidin

  • Detect MPP1 using HRP-conjugated anti-MPP1 antibodies

  • Include controls with omitted hydroxylamine treatment to verify specificity

• Metabolic labeling with palmitate analogs:

  • Incubate cells with alkyne-palmitate

  • Perform click chemistry to add detectable tags

  • Immunoprecipitate MPP1

  • Detect labeled MPP1 using HRP-conjugated anti-MPP1 antibodies

Phosphorylation analysis:

Given MPP1's role in regulating AKT1 phosphorylation in neutrophil polarization :

• Phospho-specific Western blotting:

  • Use phosphatase inhibitors during sample preparation

  • Separate samples using Phos-tag™ acrylamide gels that specifically retard phosphorylated proteins

  • Transfer and probe with HRP-conjugated anti-MPP1 antibodies

  • Compare migration patterns before and after phosphatase treatment

• Sequential immunoprecipitation approach:

  • First immunoprecipitate with anti-phosphotyrosine/serine/threonine antibodies

  • Then detect MPP1 in immunoprecipitates using HRP-conjugated anti-MPP1 antibodies

  • Alternatively, immunoprecipitate with anti-MPP1 and detect phosphorylation with phospho-specific antibodies

Ubiquitination and SUMOylation analysis:

• Denaturing immunoprecipitation:

  • Lyse cells under denaturing conditions (1% SDS, boiling)

  • Dilute and immunoprecipitate MPP1

  • Detect ubiquitin/SUMO modifications using specific antibodies

  • Verify MPP1 identity using HRP-conjugated anti-MPP1 antibodies

• Proteasome inhibitor treatment:

  • Treat cells with MG132 or other proteasome inhibitors

  • Analyze MPP1 by Western blotting using HRP-conjugated anti-MPP1 antibodies

  • Look for higher molecular weight bands indicating modification

Mass spectrometry verification:

For comprehensive PTM mapping, combine immunoprecipitation using anti-MPP1 antibodies with mass spectrometry:

• Immunoprecipitate MPP1 from relevant cell types (e.g., neutrophils or AML cells)

• Verify successful immunoprecipitation using a small portion of the sample with HRP-conjugated anti-MPP1 antibodies

• Subject the remainder to tryptic digestion and LC-MS/MS analysis

• Analyze data for mass shifts indicative of various PTMs

These approaches provide complementary information about MPP1's post-translational modification state, which may influence its interaction with ABCC4 and subsequent roles in drug resistance in AML .

What considerations should be made when designing multiplexed flow cytometry panels incorporating HRP-conjugated anti-MPP1 antibodies?

Designing multiplexed flow cytometry panels that include HRP-conjugated anti-MPP1 antibodies requires careful consideration of several technical and biological factors:

HRP detection system integration:

HRP is not typically used directly in flow cytometry due to excitation/emission limitations. Instead, researchers should consider:

• Tyramide signal amplification (TSA) approach:

  • HRP catalyzes the deposition of fluorophore-conjugated tyramide molecules

  • Select tyramide-fluorophores with emission spectra compatible with other panel fluorophores

  • Perform HRP detection first in sequential staining protocols to prevent interference

• Alternative conjugation consideration:

  • If direct detection is preferred, consider having anti-MPP1 antibodies custom-conjugated to standard flow cytometry fluorochromes instead of HRP

  • For dilute antibody preparations (<0.5 mg/mL), concentrate using an Antibody Concentration and Clean Up Kit before conjugation

Panel design for MPP1 biology:

• Cell type considerations:

  • Include markers to identify relevant MPP1-expressing populations:

    • CD45 (hematopoietic cells)

    • CD33/CD13 (myeloid markers for AML studies)

    • CD16/CD66b (neutrophil markers for polarization studies)

• Functional marker integration:

  • Include markers related to MPP1's biological functions:

    • Phospho-AKT1 (downstream of MPP1 in neutrophil polarization)

    • ABCC4 (forms functional complex with MPP1 in drug resistance)

    • Apoptosis markers (for studying drug sensitivity)

Technical optimization considerations:

• Permeabilization protocol selection:

  • MPP1 has both membrane and cytoplasmic localization

  • Test different permeabilization reagents to optimize detection while preserving epitopes

  • For studying membrane vs. cytoplasmic distribution, consider gentle permeabilization protocols

• Fixation compatibility:

  • Verify that fixation methods do not interfere with HRP activity or epitope recognition

  • Consider fixation timing (pre- vs. post-antibody staining)

• Compensation and controls:

  • Include single-stained controls for each fluorochrome

  • Use FMO (fluorescence minus one) controls to set proper gates

  • If using tyramide amplification, include controls with primary antibody omitted

Sample-specific considerations:

• AML patient samples:

  • Include viability dye to exclude dead cells

  • Consider density gradient separation to remove debris

  • Use CD34/CD38 to identify leukemic stem cell populations when studying MPP1-ABCC4 interaction

• Neutrophil polarization studies:

  • Process samples rapidly to prevent spontaneous activation

  • Consider live cell analysis where possible

  • Include polarization stimuli controls (e.g., fMLP, IL-8)

This comprehensive approach enables researchers to effectively incorporate anti-MPP1 detection into complex flow cytometry panels for studying MPP1 biology in both basic research and clinical specimens.