LAMB4 Antibody, HRP conjugated

What is LAMB4 and why is it important for research?

LAMB4 (Laminin subunit beta-4) is an extracellular matrix protein that mediates the attachment, migration, and organization of cells into tissues during embryonic development through interactions with other extracellular matrix components . Recent research has revealed that LAMB4 plays a critical role in the development of human peripheral sensory nervous system, specifically affecting the migration of sensory neuron progenitors and neural crest cells . The protein has also been identified in the myenteric plexus of the colon, with variants associated with familial diverticulitis . Understanding LAMB4's function is essential for research in neurodevelopment, sensory neuron pathologies, and gastrointestinal disorders.

What specifically does the HRP conjugation enable in LAMB4 antibody applications?

The HRP (Horseradish Peroxidase) conjugation to LAMB4 antibodies provides a direct enzymatic detection capability that eliminates the need for secondary antibody incubation steps in many applications. This conjugation enables direct visualization through chromogenic substrates (such as TMB or DAB) or chemiluminescent substrates in applications like ELISA, western blotting, and immunohistochemistry . The HRP enzyme catalyzes reactions that produce colorimetric, fluorescent, or luminescent signals proportional to the amount of target protein present, allowing for both qualitative and quantitative analyses of LAMB4 expression patterns.

What are the structural characteristics of LAMB4 that researchers should consider when selecting antibodies?

When selecting LAMB4 antibodies, researchers should consider the specific amino acid regions targeted by the antibody. Many commercially available antibodies target regions such as AA 1513-1761 or Asn1543~Cys1759 , which represent specific functional domains of the protein. LAMB4 has a calculated molecular weight of approximately 193,540 Da , which should be considered when analyzing western blot results. Additionally, LAMB4 interacts with laminin α4 and laminin γ3 to form laminin-443, a previously unreported laminin trimer . Antibodies targeting different epitopes may provide varying results based on protein conformation, trimeric assembly state, or post-translational modifications.

What are the validated applications for LAMB4 antibody, HRP conjugated, and what are the recommended working dilutions?

Based on available research, LAMB4 antibody with HRP conjugation has been validated for ELISA applications primarily , though unconjugated versions have broader application ranges. For optimal results, the recommended working dilutions are:

The optimal working dilution should be determined experimentally for each specific application and experimental condition .

How should researchers optimize immunohistochemistry protocols when studying LAMB4 in nervous tissue?

When studying LAMB4 in nervous tissue, particularly in the peripheral sensory nervous system or enteric nervous system, researchers should implement several optimization strategies:

• Tissue fixation: Paraformaldehyde (4%) fixation has been successfully used in studies localizing LAMB4 in myenteric plexus .

• Antigen retrieval: Heat-induced epitope retrieval may be necessary to unmask LAMB4 epitopes in formalin-fixed tissues.

• Blocking: Use 2% serum from the same species as the secondary antibody to reduce background .

• Co-localization markers: Include markers such as S100 (for glial cells) or Collagen IV (for epineurium) to precisely localize LAMB4 within neural structures .

• Signal visualization: For HRP-conjugated antibodies, optimize DAB development time (typically 2-10 minutes) to achieve optimal signal-to-noise ratio.

• Validation controls: Include tissue sections stained with only primary or secondary antibodies to confirm specificity .

LAMB4 has been shown to localize specifically to the endoneurium of the myenteric plexus rather than the epineurium, which should be considered when interpreting results .

How should researchers approach experimental design when studying LAMB4 expression changes in developmental models?

When studying LAMB4 expression changes during development, particularly in the context of peripheral sensory nervous system development, researchers should consider:

• Temporal sampling: Establish a timeline of collection points that captures key developmental stages, particularly during neural crest migration and sensory neuron specification .

• Control selection: Include appropriate developmental stage-matched controls and consider using CRISPR/Cas9 LAMB4 knockout models as negative controls .

• Model systems: Human pluripotent stem cells (hPSCs) have been successfully used to study LAMB4's role in sensory neuron development . Consider using these models for in vitro developmental studies.

• Quantification methods: Employ multiple quantification strategies including:

  • Protein level assessment using western blot with appropriate loading controls

  • Transcript level analysis using qRT-PCR with validated reference genes

  • Spatial distribution analysis using immunohistochemistry with digital image analysis

• Functional assays: Include migration assays for neural crest cells to assess LAMB4's functional impact on development .

• Statistical planning: Design experiments with sufficient biological and technical replicates to achieve statistical power, particularly when studying subtle developmental phenotypes.

What approaches can be used to validate LAMB4 antibody specificity in experimental systems?

Validating LAMB4 antibody specificity is critical for experimental rigor. Researchers should implement multiple validation strategies:

• Genetic knockdown/knockout controls: Use CRISPR/Cas9-mediated LAMB4 knockout cells or tissues as negative controls .

• Overexpression systems: Test antibody reactivity in cells transiently transfected with a plasmid expressing LAMB4 cDNA versus vector-only transfected cells .

• Peptide competition assays: Pre-incubate the antibody with the immunizing peptide to demonstrate signal reduction.

• Multiple antibody validation: Use antibodies from different suppliers or targeting different epitopes to confirm staining patterns.

• Cross-reactivity testing: Assess potential cross-reactivity with related laminin family proteins, particularly other beta subunits.

• Primary and secondary antibody controls: Include controls lacking primary or secondary antibody to assess non-specific binding .

• Technical validation: Confirm antibody reactivity across multiple applications (ELISA, WB, IHC) to ensure consistent target recognition.

What are common troubleshooting strategies when LAMB4 antibody, HRP conjugated shows weak or inconsistent signal?

When encountering weak or inconsistent signals with LAMB4 antibody, HRP conjugated, consider these troubleshooting approaches:

• Antibody concentration: Titrate the antibody concentration, starting with the manufacturer's recommended dilution and then adjusting as necessary. For HRP-conjugated antibodies, a dilution series between 1:1000-1:20000 should be tested .

• Sample preparation: Ensure proper sample preparation, including:

  • For tissue samples: Optimize fixation time and conditions

  • For protein samples: Verify protein extraction efficiency and prevent proteolytic degradation with appropriate inhibitors

• Storage and handling: HRP-conjugated antibodies should be stored at -20°C or -80°C and avoid repeated freeze-thaw cycles . Use glycerol-containing buffers (typically 50% glycerol) to prevent freezing damage .

• Antigen accessibility: For tissue sections or fixed cells, optimize antigen retrieval methods (heat-induced or enzymatic).

• Detection system: For weak signals, consider enhanced chemiluminescence (ECL) substrates with higher sensitivity or signal amplification methods.

• Blocking conditions: Adjust blocking reagents (BSA, normal serum, commercial blockers) and duration to reduce background while preserving specific signal.

• Buffer conditions: Verify the pH and ionic strength of wash and incubation buffers, as LAMB4 antibody performance may be affected by these parameters .

How should researchers analyze and interpret contradictory LAMB4 expression data between different experimental approaches?

When faced with contradictory LAMB4 expression data between different experimental approaches, researchers should follow this analytical framework:

• Technical assessment:

  • Evaluate antibody specificity in each application using appropriate controls

  • Confirm that proper normalization was applied for quantitative methods

  • Assess whether differences in sample preparation could explain the discrepancies

• Biological considerations:

  • Determine if discrepancies might reflect actual biological differences in LAMB4 isoforms, post-translational modifications, or protein-protein interactions

  • Consider that LAMB4 forms a complex with laminin α4 and laminin γ3 (laminin-443) , which may affect epitope accessibility

• Method-specific limitations:

  • Western blot: May detect denatured epitopes not accessible in fixed tissues

  • IHC/ICC: May be affected by fixation-induced epitope masking

  • ELISA: May detect soluble forms or fragments not visible by other methods

• Resolution strategies:

  • Employ additional orthogonal methods (e.g., mRNA analysis, mass spectrometry)

  • Use multiple antibodies targeting different LAMB4 epitopes

  • Conduct genetic validation (siRNA knockdown, CRISPR knockout)

  • Perform domain-specific analyses to determine if contradictions reflect domain-specific effects

• Data integration approach:

  • Weight evidence based on methodological rigor and control quality

  • Consider biological context when interpreting discrepancies

  • Report contradictions transparently in publications

How can LAMB4 antibody, HRP conjugated be utilized in co-localization studies examining extracellular matrix assembly in sensory neuron development?

For co-localization studies examining extracellular matrix assembly in sensory neuron development, LAMB4 antibody (HRP conjugated) can be integrated into multiplexed imaging approaches:

• Sequential chromogenic detection: When using HRP-conjugated LAMB4 antibody:

  • First detect LAMB4 using HRP substrate (e.g., DAB)

  • Apply hydrogen peroxide to inactivate the first HRP reaction

  • Apply second primary antibody (e.g., against laminin α4 or γ3)

  • Detect with a different chromogen (e.g., AEC, producing red color)

• Combined fluorescent and chromogenic detection:

  • Use tyramide signal amplification (TSA) to convert HRP signal to a fluorescent signal

  • Combine with fluorescently-labeled antibodies against other ECM components

• Multi-round staining approach:

  • Strip and reprobe membranes or slides after LAMB4 detection

  • Use digital image registration to align and analyze multiple staining rounds

• Relevant co-localization partners:

  • Laminin α4 and γ3 to study laminin-443 trimer formation

  • Actin filaments to study LAMB4's role in cytoskeletal organization

  • Neural crest markers (SOX10, p75NTR) to study LAMB4's role in neural crest cell migration

  • Sensory neuron markers (TRKA, TRKB, TRKC) to study LAMB4's impact on specific sensory neuron subtypes

• Analytical approaches:

  • Calculate Pearson's or Mander's correlation coefficients between LAMB4 and other proteins

  • Perform distance mapping to quantify spatial relationships between LAMB4 and cellular structures

What are the most sensitive methods for detecting subtle alterations in LAMB4 expression in patient-derived iPSC models of neurological disorders?

For detecting subtle alterations in LAMB4 expression in patient-derived iPSC models of neurological disorders, researchers should consider these high-sensitivity approaches:

• Digital droplet PCR (ddPCR) for transcript quantification:

  • Provides absolute quantification with higher precision than qRT-PCR

  • Less susceptible to PCR inhibitors that may be present in complex samples

  • Particularly useful for detecting small fold-changes (1.2-1.5x) that might be biologically relevant

• Mass spectrometry-based proteomics:

  • Targeted approaches such as parallel reaction monitoring (PRM) or selected reaction monitoring (SRM)

  • Label-free quantification with high-resolution instruments

  • SILAC or TMT labeling for precise relative quantification across samples

• Enhanced immunodetection methods:

  • Tyramide signal amplification (TSA) to enhance sensitivity of HRP-conjugated antibodies

  • Proximity ligation assay (PLA) to detect protein-protein interactions involving LAMB4

  • Ultrasensitive ELISA with amplification steps

• Single-cell analysis approaches:

  • Single-cell RNA-seq to identify cell populations with altered LAMB4 expression

  • Mass cytometry (CyTOF) with metal-conjugated antibodies

  • Spatial transcriptomics to map LAMB4 expression changes in complex tissues

• Live-cell imaging techniques:

  • CRISPR-based endogenous tagging of LAMB4 with fluorescent proteins

  • Antibody internalization assays to track dynamics of LAMB4 processing

Recent research has demonstrated that human pluripotent stem cells (hPSCs) and induced pluripotent stem cells (iPSCs) can be effectively used to study LAMB4's role in sensory neuron development and neurological conditions like Familial Dysautonomia , making these approaches particularly relevant for translational neurological research.