LAMA2 Antibody, HRP conjugated

What is LAMA2 and what cellular functions does it mediate?

LAMA2 (Laminin subunit alpha-2) is a large protein (approximately 343.9 kDa) that functions as an extracellular matrix structural constituent and participates in signaling receptor binding . The protein consists of 3122 amino acids and plays critical roles in development, extracellular matrix organization, and signal transduction pathways . LAMA2 is also known by several synonyms including Laminin M chain, Laminin-12 subunit alpha, Laminin-2 subunit alpha, Laminin-4 subunit alpha, and Merosin heavy chain . This protein undergoes post-translational modifications, most notably glycosylation, which can impact its functional properties and detection methods .

What are the structural characteristics of LAMA2 antibodies, and specifically HRP-conjugated variants?

LAMA2 antibodies are immunoglobulins raised against specific epitopes of the Laminin subunit alpha-2 protein. The HRP-conjugated variants have horseradish peroxidase enzyme covalently attached to the antibody structure, enabling direct detection without secondary antibodies . Typical LAMA2 antibodies used in research are often polyclonal IgGs raised in rabbits, purified through methods such as Protein G purification with purity levels exceeding 95% . When conjugated with HRP, these antibodies are typically supplied in a liquid form with storage buffers containing preservatives (0.03% Proclin 300), stabilizers (50% Glycerol), and buffering agents (0.01M PBS, pH 7.4) .

What are the typical storage requirements for maintaining LAMA2 antibody, HRP conjugated activity?

For optimal preservation of enzymatic activity and binding specificity, LAMA2 antibodies with HRP conjugation should be stored at -20°C or -80°C immediately upon receipt . Repeated freeze-thaw cycles must be strictly avoided as they can significantly degrade both the antibody binding capacity and the HRP enzymatic activity . For shipping purposes, these antibodies are typically transported at 4°C, but long-term storage at this temperature is not recommended . Many suppliers provide these antibodies in 50% glycerol buffer, which acts as a cryoprotectant to minimize damage during the freezing process .

How should researchers select the appropriate immunogen region when evaluating LAMA2 antibodies for specific applications?

When selecting LAMA2 antibodies, researchers should carefully evaluate the immunogen used in antibody production, as this directly impacts epitope recognition and application performance . For HRP-conjugated LAMA2 antibodies, several commercial options utilize recombinant human Laminin subunit alpha-2 protein fragments, such as the region spanning amino acids 974-1162, which has demonstrated reliable detection in ELISA applications . For applications requiring detection of different protein domains, researchers should consider antibodies raised against alternative regions, such as the synthetic peptide derived from amino acids 2011-2060, which has shown efficacy in immunohistochemistry and immunofluorescence . Researchers working with specific LAMA2 isoforms or seeking to detect post-translational modifications should select antibodies with immunogens that encompass these regions of interest .

What are the optimized dilution protocols for LAMA2 antibody, HRP conjugated across different experimental applications?

Optimal dilution protocols vary significantly based on the specific application and detection system employed. For ELISA applications using HRP-conjugated LAMA2 antibodies, dilutions ranging from 1:5,000 to 1:20,000 have demonstrated suitable signal-to-noise ratios . For applications requiring higher sensitivity or when working with scarce samples, researchers should begin with more concentrated solutions (1:5,000) and optimize based on signal intensity and background levels . For immunohistochemistry applications, significantly more concentrated solutions (1:50 to 1:100) are typically required to achieve adequate tissue staining . For immunofluorescence, intermediate dilutions (1:100 to 1:500) generally provide optimal results . These dilution recommendations should serve as starting points, with further optimization necessary for specific experimental conditions, tissue types, and detection systems.

What sample preparation techniques maximize LAMA2 epitope accessibility when using HRP-conjugated antibodies?

Effective sample preparation is critical for maximizing epitope accessibility and achieving specific LAMA2 detection . For tissue sections used in immunohistochemistry, heat-induced epitope retrieval (HIER) using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0) is recommended to reverse formalin-induced protein cross-linking that might mask LAMA2 epitopes . When working with cell lysates for ELISA applications, complete solubilization using buffers containing non-ionic detergents (0.5-1% Triton X-100 or NP-40) helps expose LAMA2 epitopes while preserving antibody-binding capacity . For extracellular matrix proteins like LAMA2, enzymatic pre-treatment with hyaluronidase (10-20 minutes at 37°C) may significantly improve antibody penetration and epitope recognition . Additionally, blocking with 5% BSA or normal serum from the same species as the secondary antibody (if used in a detection cascade) reduces non-specific binding and improves signal-to-noise ratio .

What are the expected molecular weight variations for LAMA2 detection across different experimental systems?

LAMA2 has a theoretical molecular weight of approximately 343.9 kDa based on its amino acid composition (3122 amino acids), though observed molecular weights may vary in experimental systems . In Western blot applications, researchers may observe bands ranging from 340-350 kDa for the full-length protein . Post-translational modifications, particularly glycosylation, can increase the apparent molecular weight by 5-10% . Additionally, LAMA2 undergoes proteolytic processing, potentially generating fragments of approximately 300 kDa (N-terminal fragment) and 80 kDa (C-terminal fragment) that may be detected depending on the epitope recognized by the antibody . When analyzing samples from different species, minor variations in molecular weight may be observed due to sequence differences, though the protein remains highly conserved between human and mouse models .

How can researchers distinguish between specific and non-specific binding when using LAMA2 antibody, HRP conjugated?

Distinguishing specific from non-specific binding is crucial for accurate data interpretation when using HRP-conjugated LAMA2 antibodies . Implementing proper controls is essential: negative controls should include samples known to lack LAMA2 expression, while isotype controls using non-specific IgG (matched to the LAMA2 antibody's host species and isotype) help identify background signal from non-specific binding . For tissue analysis, parallel sections stained with secondary antibody only (for detection systems using secondary antibodies) can reveal background issues . Signal specificity can be further validated through peptide competition assays, where pre-incubation of the antibody with the immunizing peptide should substantially reduce or eliminate specific binding . Additionally, comparing staining patterns across multiple LAMA2 antibodies targeting different epitopes can confirm signal specificity, as true positive signals should show consistent localization patterns .

What are the most common causes of high background when using HRP-conjugated LAMA2 antibodies, and how can they be mitigated?

High background is a common challenge when working with HRP-conjugated antibodies, including those targeting LAMA2 . Several factors can contribute to this issue:

• Insufficient blocking: Inadequate blocking leads to non-specific binding. Mitigation involves extending blocking time (1-2 hours) and optimizing blocker concentration (3-5% BSA or normal serum) .

• Excessive antibody concentration: Too high antibody concentrations increase non-specific interactions. This can be resolved by performing titration experiments to identify the minimum concentration needed for specific detection .

• Sample fixation artifacts: Overfixation can create reactive groups that bind antibodies non-specifically. Using freshly prepared fixatives and optimizing fixation duration helps minimize these artifacts .

• Endogenous peroxidase activity: Tissues, particularly those rich in erythrocytes, contain endogenous peroxidases that react with HRP substrates. Pre-treatment with 0.3% H₂O₂ in methanol for 10-30 minutes effectively quenches this activity .

• Biotin interference: For detection systems involving biotin, endogenous biotin can create false signals. Using biotin-free detection systems or incorporating an avidin/biotin blocking step resolves this issue .

• Cross-reactivity: Antibody cross-reactivity with structurally similar proteins can be minimized by selecting highly validated antibodies and confirming specificity through knockout or knockdown controls .

How can LAMA2 antibody, HRP conjugated be effectively utilized in multiplex immunoassays with other ECM protein markers?

For advanced multiplex assays combining LAMA2 detection with other extracellular matrix proteins, researchers can implement several sophisticated strategies . When designing multiplex ELISA panels, the HRP-conjugated LAMA2 antibody can be paired with antibodies conjugated to different reporter enzymes (such as alkaline phosphatase) that utilize distinct chromogenic or fluorogenic substrates with non-overlapping emission spectra . For tissue-based multiplexing, sequential immunostaining protocols can be employed, where complete development and quenching of the HRP signal from the LAMA2 antibody precedes application of subsequent antibodies . Tyramide signal amplification (TSA) systems are particularly valuable in this context, as they allow the deposition of spectrally distinct fluorophores at LAMA2 binding sites, followed by heat-mediated antibody stripping before the next staining round . Researchers should carefully validate each antibody combination to ensure that antibody stripping does not affect tissue morphology or antigen preservation for subsequent detection steps .

What are the considerations for using LAMA2 antibody, HRP conjugated in research on merosin-deficient congenital muscular dystrophy (MDC1A)?

LAMA2 mutations are causally linked to merosin-deficient congenital muscular dystrophy (MDC1A), making LAMA2 antibodies valuable tools in studying this condition . When designing experiments for MDC1A research, several methodological considerations are critical. First, epitope selection is paramount—antibodies recognizing epitopes within commonly mutated regions may yield false-negative results in patient samples . Researchers should select antibodies recognizing conserved epitopes or use multiple antibodies targeting different domains . Second, quantitative analysis of LAMA2 expression requires careful standardization against housekeeping proteins or total protein stains, with consistent imaging parameters across specimens . Third, for distinguishing partial from complete LAMA2 deficiency, titration studies with serial antibody dilutions provide enhanced sensitivity to detect low-level expression . Finally, correlation of immunohistochemical findings with functional assays measuring basement membrane integrity and muscle fiber attachment strength provides more comprehensive insights into pathophysiological mechanisms .

How can researchers effectively combine LAMA2 antibody detection with laser capture microdissection for region-specific ECM analysis?

Integrating LAMA2 immunodetection with laser capture microdissection (LCM) requires specialized protocols to maintain both antigen recognition and RNA/protein integrity for downstream analysis . The most effective approach involves a modified rapid immunostaining protocol: fixing tissue sections briefly (5-10 minutes) in ice-cold 70-80% ethanol rather than formalin, blocking for a shortened duration (10 minutes), and using higher concentrations of HRP-conjugated LAMA2 antibody (approximately 2-5× standard IHC concentration) with reduced incubation times (15-30 minutes) . RNase inhibitors should be incorporated into all solutions if RNA analysis is planned post-microdissection . For the detection step, DAB development should be carefully monitored and minimized (15-90 seconds) to prevent excessive precipitation that might interfere with laser cutting precision . Following microdissection of LAMA2-positive regions, researchers can extract proteins using specialized buffers compatible with downstream proteomics workflows, typically containing chaotropic agents (6-8M urea), reducing agents, and protease inhibitors . This approach enables correlation between LAMA2 distribution patterns and comprehensive proteomic profiles of the surrounding extracellular matrix microenvironment .

What methodological approaches enable reliable quantification of LAMA2 levels in complex tissue microenvironments?

Quantifying LAMA2 in complex tissues requires sophisticated approaches that account for tissue heterogeneity and complex protein distribution patterns . Digital image analysis using supervised machine learning algorithms can be trained to recognize LAMA2-positive structures based on morphological features and staining intensity, enabling automated quantification across entire tissue sections . For more precise quantification, multiplexed immunofluorescence combining LAMA2 detection with cell-type-specific markers allows normalization of LAMA2 signals to defined cellular compartments or tissue regions . When absolute quantification is required, researchers should implement calibrated measurement systems using tissue microarrays containing standards with known LAMA2 concentrations processed identically to experimental samples . For three-dimensional analysis of LAMA2 distribution, tissue clearing techniques (CLARITY, CUBIC, or iDISCO) combined with whole-mount immunolabeling using HRP-conjugated antibodies and tyramide signal amplification enable volumetric assessment of LAMA2 networks through confocal or light-sheet microscopy . These advanced quantification approaches significantly enhance the ability to detect subtle changes in LAMA2 expression patterns associated with pathological processes .

What strategies can be implemented to optimize signal-to-noise ratio when using LAMA2 antibody, HRP conjugated in challenging tissue types?

Optimizing signal-to-noise ratio for LAMA2 detection in challenging tissues (such as fibrotic tissues or those with high autofluorescence) requires methodological refinements beyond standard protocols . For tissues with dense extracellular matrix components that might impede antibody penetration, pre-treatment with hyaluronidase (100 U/ml) followed by brief proteinase K digestion (5-10 µg/ml for 5-10 minutes) significantly enhances epitope accessibility without destroying tissue architecture . When using HRP-conjugated antibodies in tissues with high endogenous peroxidase activity (like liver or kidney), a dual quenching approach using 0.3% H₂O₂ followed by 0.1% sodium azide treatment provides superior background reduction compared to standard methods . For highly autofluorescent tissues, Sudan Black B (0.1-0.3% in 70% ethanol) applied post-immunostaining effectively quenches lipofuscin-derived autofluorescence . Signal amplification using tyramide-based systems can enhance specific signal detection by 10-50 fold while maintaining spatial resolution, particularly valuable for detecting low LAMA2 expression in pathological samples . For each challenging tissue type, researchers should implement a systematic optimization matrix varying antigen retrieval conditions, antibody concentration, incubation time, and detection methods to identify the optimal protocol combination .

How can orthogonal validation techniques confirm the specificity of findings obtained with LAMA2 antibody, HRP conjugated?

• Genetic validation: Testing antibody reactivity in LAMA2 knockout/knockdown models or using siRNA-mediated LAMA2 silencing in cell culture systems provides definitive confirmation of specificity .

• Recombinant protein controls: Parallel analysis of recombinant LAMA2 protein alongside experimental samples can verify correct molecular weight detection and antibody functionality .

• Alternative antibody comparison: Using multiple LAMA2 antibodies targeting different epitopes should yield consistent localization patterns if detection is specific .

• Transcriptional correlation: Correlating protein detection with mRNA expression through RNA-seq or qPCR provides additional validation of expression patterns .

• Mass spectrometry verification: Immunoprecipitation using the LAMA2 antibody followed by mass spectrometry analysis can definitively identify the captured proteins and confirm LAMA2 enrichment .

• Cross-species reactivity assessment: Testing antibody performance across species with known sequence conservation or divergence helps establish specificity boundaries .

These orthogonal approaches collectively build a robust validation framework that substantially increases confidence in experimental findings .

How can LAMA2 antibody, HRP conjugated be effectively utilized in single-cell protein analysis techniques?

Adapting LAMA2 antibodies for single-cell protein analysis represents an emerging frontier in extracellular matrix research . For mass cytometry (CyTOF) applications, HRP-conjugated LAMA2 antibodies can be modified through metal chelation, where lanthanide metals are attached to the antibody via the HRP moiety, enabling highly multiplexed single-cell analysis with minimal spectral overlap . In microfluidic-based single-cell western blotting, the high sensitivity of HRP-based chemiluminescent detection is particularly valuable for quantifying LAMA2 expression in individual cells sorted from heterogeneous populations . For in situ analysis with subcellular resolution, proximity ligation assays (PLA) using LAMA2 antibodies in combination with antibodies against potential binding partners can visualize specific protein-protein interactions at the single-molecule level . Recent advances in spatial proteomics platforms, such as Digital Spatial Profiling (DSP) and CODEX, can incorporate HRP-conjugated LAMA2 antibodies to generate spatially resolved protein expression maps at near-single-cell resolution . These advanced techniques enable researchers to investigate cell-specific variations in LAMA2 expression and interactions that would be masked in bulk tissue analyses .

What considerations are important when using LAMA2 antibody, HRP conjugated in organ-on-chip or 3D cell culture systems?

Three-dimensional culture systems and organ-on-chip platforms present unique challenges for LAMA2 immunodetection that require specialized approaches . Antibody penetration is a primary concern in dense 3D structures—researchers should implement extended incubation periods (24-48 hours at 4°C) with gentle agitation and higher antibody concentrations (2-3× those used for 2D cultures) . For whole-mount immunostaining of organoids or microtissues, lipid clearing methods such as CUBIC or CLARITY significantly improve antibody access to internal structures while preserving LAMA2 epitopes . In perfusable organ-on-chip systems, microfluidic delivery of antibodies under controlled flow rates (typically 1-5 μL/min) can enhance uniform distribution while minimizing shear stress-induced artifacts . For quantitative analysis, confocal microscopy with deconvolution or light-sheet microscopy provides superior 3D visualization of LAMA2 distribution throughout complex structures . When analyzing LAMA2 deposition during extracellular matrix formation in these systems, time-course experiments with pulse-chase labeling strategies can differentiate newly synthesized from pre-existing LAMA2 networks . These methodological adaptations enable researchers to investigate LAMA2 dynamics in physiologically relevant 3D microenvironments that better recapitulate in vivo conditions .

How can computational image analysis enhance quantitative assessment of LAMA2 distribution patterns detected with HRP-conjugated antibodies?

Advanced computational approaches significantly enhance the extraction of quantitative information from LAMA2 immunostaining data . Deep learning-based segmentation algorithms can now accurately distinguish LAMA2-positive basement membrane structures from surrounding tissue with performance approaching expert human annotation . These algorithms can be trained on manually annotated subsets of images and then applied to large image datasets, enabling high-throughput analysis of LAMA2 distribution patterns . Texture analysis algorithms quantify the organizational properties of LAMA2 networks, measuring parameters such as fiber alignment, network connectivity, and structural periodicity that may be altered in pathological states . For co-localization studies, object-based colocalization analysis rather than pixel-based methods provides more biologically meaningful measures of spatial relationships between LAMA2 and other proteins . When analyzing time-series data, optical flow algorithms can track dynamic changes in LAMA2 deposition and remodeling . Integration of imaging data with transcriptomic or proteomic datasets through multimodal data fusion approaches enables systems-level analysis of LAMA2 regulation and function . Implementation of these computational approaches requires interdisciplinary collaboration but greatly enhances the quantitative rigor and reproducibility of LAMA2 research .

What are the emerging technological developments that will enhance LAMA2 detection sensitivity and specificity in the near future?

Several technological innovations are poised to transform LAMA2 detection capabilities in coming years . Engineered antibody fragments (nanobodies, single-domain antibodies) with superior tissue penetration properties are being developed against LAMA2 epitopes, potentially offering enhanced access to sterically hindered basement membrane regions . CRISPR-based techniques for endogenous LAMA2 tagging will enable live-cell imaging of LAMA2 dynamics without antibody-based detection, circumventing traditional limitations of fixation and permeabilization . Advances in non-destructive imaging technologies, including label-free techniques such as stimulated Raman scattering microscopy, may enable visualization of LAMA2 and other ECM components based on their intrinsic molecular vibration signatures . Next-generation spatially resolved proteomics approaches, including high-definition spatial transcriptomics integrated with in situ protein detection, will provide unprecedented insights into LAMA2 expression regulation at the single-cell level within tissue contexts . These technological developments will collectively advance our understanding of LAMA2's complex roles in tissue development, homeostasis, and disease pathogenesis .