MEGF10 Antibody, Biotin conjugated

What is MEGF10 and what cellular functions does it perform?

MEGF10 (Multiple EGF-like domains protein 10) is a transmembrane protein primarily localized to the cell membrane that mediates several critical cellular processes. It plays a significant role in phagocytosis by macrophages, specifically in the clearance of apoptotic cells. MEGF10 cooperates with ABCA1 during engulfment and destabilizes the oligomeric assemblies of the ABCA1 transporter. Additionally, it functions in cell adhesion while inhibiting cell motility and proliferation in vitro. Research has shown that MEGF10 promotes the formation of large intracellular vacuoles, suggesting its involvement in cellular trafficking mechanisms . Understanding these functions is essential for designing experiments that effectively target MEGF10-mediated processes.

What applications are biotin-conjugated MEGF10 antibodies suitable for?

Biotin-conjugated MEGF10 antibodies are primarily validated for Western Blot (WB) applications with recommended dilutions ranging from 1:300 to 1:5000 . The biotin conjugation provides enhanced sensitivity through the strong biotin-streptavidin interaction, making these antibodies particularly useful for detecting low-abundance MEGF10 protein. While WB is the primary validated application, researchers should note that unconjugated versions of MEGF10 antibodies have been validated for additional applications including immunofluorescence (IF), immunohistochemistry (IHC), and ELISA . For optimal results, preliminary titration experiments should be conducted to determine the optimal antibody concentration for your specific experimental system.

How should biotin-conjugated MEGF10 antibodies be stored to maintain reactivity?

For long-term storage, biotin-conjugated MEGF10 antibodies should be stored at -20°C, where they typically remain stable for up to 12 months . For short-term storage and frequent use, the antibodies can be kept at 4°C for up to one month . The storage buffer typically contains 0.01M TBS (pH 7.4) with 1% BSA, 0.03% Proclin300, and 50% Glycerol, which helps maintain antibody stability . Repeated freeze-thaw cycles should be avoided as they can compromise antibody activity. When using the antibody, aliquoting into smaller volumes for single-use applications is recommended to prevent repeated freeze-thaw damage to the stock solution.

What is the species reactivity profile of biotin-conjugated MEGF10 antibodies?

The biotin-conjugated MEGF10 polyclonal antibody demonstrates confirmed reactivity with human MEGF10 protein . Additionally, predicted reactivity extends to multiple species including mouse, rat, cow, sheep, pig, horse, chicken, and rabbit, though these cross-reactivities may require validation for specific experimental systems . When planning cross-species experiments, it is advisable to first confirm reactivity in your species of interest through a preliminary Western blot before proceeding with more complex experimental designs. The broad cross-reactivity profile makes these antibodies potentially valuable for comparative studies across species.

How can I validate the specificity of biotin-conjugated MEGF10 antibodies in my experimental system?

To rigorously validate antibody specificity, a multi-step approach is recommended. Begin with a Western blot analysis using positive controls (tissues or cell lines known to express MEGF10) alongside negative controls (MEGF10 knockout tissues/cells if available). Observe for a single band at the expected molecular weight of approximately 122 kDa . For more stringent validation, perform peptide competition assays where pre-incubation of the antibody with the immunizing peptide should abolish signal. Additionally, consider using siRNA knockdown of MEGF10 in your experimental system to confirm specificity. For biotin-conjugated antibodies specifically, include controls to rule out non-specific binding of the streptavidin detection system by running samples with streptavidin alone. Document these validation steps thoroughly to support the reliability of your experimental findings.

What are the optimal conditions for using biotin-conjugated MEGF10 antibodies in Western blot applications?

For optimal Western blot results with biotin-conjugated MEGF10 antibodies, begin with sample preparation that preserves the native epitope. MEGF10 is a membrane protein, so effective membrane protein extraction protocols are essential. Use dilutions in the range of 1:300 to 1:5000, with initial optimization experiments recommended . For detection, streptavidin-HRP conjugates are typically used, with careful titration to minimize background. When probing for MEGF10, use 3-5% BSA for blocking rather than milk, as milk can contain biotin that may interfere with detection. For membrane proteins like MEGF10, sample heating should be limited to 70°C for 5 minutes rather than boiling, which can cause aggregation. Additionally, the use of PVDF membranes rather than nitrocellulose may improve detection of this higher molecular weight protein (122 kDa) .

How can I troubleshoot weak or absent signals when using biotin-conjugated MEGF10 antibodies?

When encountering weak or absent signals, consider several methodological adjustments. First, verify protein expression in your samples using alternative MEGF10 antibodies or RT-PCR. Increase protein loading (50-100 μg total protein) as MEGF10 may be expressed at low levels in some tissues. For biotin-conjugated antibodies specifically, ensure your detection system (streptavidin-HRP) is functional by including a biotinylated protein control. Consider extending primary antibody incubation to overnight at 4°C and increasing antibody concentration. For membrane proteins like MEGF10, incomplete protein transfer can be an issue; verify transfer efficiency with reversible protein stains before immunodetection. If signal remains weak, signal amplification systems compatible with biotin-streptavidin detection can be employed. Finally, ensure the epitope is not masked by sample preparation methods or post-translational modifications by testing alternative protein extraction and denaturation protocols.

How can biotin-conjugated MEGF10 antibodies be integrated into multi-color flow cytometry or immunofluorescence protocols?

Integrating biotin-conjugated MEGF10 antibodies into multi-color protocols requires careful consideration of fluorophore selection and sequential staining approaches. Begin by using streptavidin conjugated to a fluorophore with minimal spectral overlap with your other detection channels (common options include streptavidin-PE, streptavidin-APC, or streptavidin-Alexa Fluor 647). For multi-color immunofluorescence, apply the biotin-conjugated MEGF10 antibody first, followed by streptavidin-fluorophore, then proceed with additional primary and secondary antibody pairs. This sequential approach prevents cross-reactivity between detection systems. When designing multi-color panels, account for MEGF10's membrane localization when interpreting co-localization with other proteins . For flow cytometry applications, thorough controls including FMO (fluorescence minus one) with the biotin-conjugated antibody are essential to accurately set gates and compensate for any biotin-streptavidin background.

What experimental approaches can resolve contradictory results when studying MEGF10 function using biotin-conjugated antibodies?

When facing contradictory results, implement a systematic approach to identify potential sources of variability. First, verify antibody lot consistency by requesting Certificate of Analysis documents from the manufacturer. Consider epitope differences between antibody clones – the biotin-conjugated MEGF10 antibody targets the region spanning amino acids 1041-1140 , which may behave differently than antibodies targeting other regions. Employ multiple detection methods, combining the biotin-conjugated antibody approach with unconjugated MEGF10 antibodies or orthogonal techniques like mass spectrometry. For functional studies, complement antibody-based detection with genetic approaches such as CRISPR-Cas9 knockout or knockdown. Cell-specific and context-dependent functions of MEGF10 should be considered, as MEGF10 has multiple roles in different cell types, including phagocytosis in macrophages and cell adhesion functions . Finally, assess potential interference from endogenous biotin in your experimental system, which can be blocked using avidin-biotin blocking kits.

How can I quantitatively assess MEGF10 expression levels in different cell states using biotin-conjugated antibodies?

For quantitative assessment of MEGF10 expression, implement a systematic approach combining multiple methodologies. For Western blot quantification, use validated loading controls appropriate for membrane proteins, such as Na+/K+ ATPase rather than common cytosolic proteins like GAPDH or β-actin. Develop standard curves using recombinant MEGF10 protein of known concentration to enable absolute quantification. For immunofluorescence quantification, utilize automated image analysis software with appropriate cell segmentation algorithms to measure membrane-localized MEGF10 signal intensity . When comparing different cell states, simultaneous processing and analysis of all samples is crucial to minimize technical variability. Additionally, validate protein-level quantification with mRNA analysis through qRT-PCR. For more precise quantification, consider implementing stable isotope labeling methods combined with immunoprecipitation using the biotin-conjugated antibody followed by mass spectrometry analysis, which can provide absolute quantification of MEGF10 protein levels across different experimental conditions.

What controls should be included when using biotin-conjugated MEGF10 antibodies in co-immunoprecipitation experiments?

When designing co-immunoprecipitation experiments with biotin-conjugated MEGF10 antibodies, a comprehensive set of controls is essential. Include an isotype control (biotin-conjugated rabbit IgG) processed identically to your experimental samples to identify non-specific binding. Additionally, incorporate a pre-clearing step using streptavidin beads alone to remove endogenously biotinylated proteins. A critical negative control should include immunoprecipitation from cells where MEGF10 has been knocked down or knocked out. For analyzing MEGF10's reported interaction with ABCA1 , include reciprocal co-immunoprecipitation using anti-ABCA1 antibodies. To control for potential biotin interference, compare results between biotin-conjugated and unconjugated MEGF10 antibodies targeting the same epitope. When blotting co-immunoprecipitated complexes, use antibodies raised in species different from the immunoprecipitating antibody to avoid detection of heavy and light chains. Finally, validate interactions using additional approaches such as proximity ligation assays or FRET to confirm direct protein-protein associations.

How can biotin-conjugated MEGF10 antibodies be used to investigate its role in phagocytosis of apoptotic cells?

To investigate MEGF10's role in phagocytosis, design experiments that visualize the protein during distinct phases of the phagocytic process. Begin with immunofluorescence studies using biotin-conjugated MEGF10 antibodies followed by fluorescent streptavidin to track MEGF10 localization during phagocytosis. Implement live-cell imaging with fluorescently labeled apoptotic cells (using pHrodo or similar pH-sensitive dyes) to correlate MEGF10 recruitment with engulfment events. Given MEGF10's known cooperation with ABCA1 during engulfment , design co-localization studies to visualize both proteins during phagocytosis using multi-color immunofluorescence. For functional validation, compare phagocytic efficiency in wild-type versus MEGF10-depleted cells using flow cytometry-based phagocytosis assays. Additionally, employ structure-function studies using truncation mutants of MEGF10 in reconstitution experiments to identify which domains are essential for phagocytic activity. For mechanistic studies, investigate how MEGF10 destabilizes ABCA1 oligomeric assemblies using techniques such as blue native PAGE combined with Western blotting using the biotin-conjugated antibody.

What methodological approaches can assess MEGF10's impact on cell motility and proliferation using biotin-conjugated antibodies?

To investigate MEGF10's reported inhibitory effects on cell motility and proliferation , implement a multi-faceted experimental approach. For cell motility studies, conduct wound healing assays comparing migration rates between cells with normal and manipulated MEGF10 expression, using the biotin-conjugated antibody to confirm expression levels. Complement this with single-cell tracking in 2D and 3D matrices, correlating movement parameters with MEGF10 expression levels quantified via immunofluorescence. For proliferation analysis, combine EdU incorporation assays with MEGF10 immunostaining to directly correlate proliferation status with MEGF10 expression at the single-cell level. To investigate underlying mechanisms, use the biotin-conjugated antibody in conjunction with phospho-specific antibodies against key proliferation and motility regulators to identify signaling pathways affected by MEGF10. Proximity ligation assays can reveal direct interactions between MEGF10 and motility/proliferation regulatory proteins. Finally, rescue experiments where wild-type or mutant MEGF10 is re-expressed in knockout cells can definitively establish causality between MEGF10 and the observed phenotypes. Throughout these experiments, quantitative image analysis should be employed to extract numerical data for statistical analysis.

How should researchers approach epitope masking issues when using biotin-conjugated MEGF10 antibodies?

When dealing with potential epitope masking of MEGF10, implement a systematic troubleshooting approach. The biotin-conjugated MEGF10 antibody targets amino acids 1041-1140 , so consider whether this region might be obscured by protein-protein interactions, post-translational modifications, or conformational changes in your experimental context. Test multiple antigen retrieval methods for fixed samples, including heat-induced epitope retrieval with citrate buffer (pH 6.0) and Tris-EDTA buffer (pH 9.0), as well as enzymatic retrieval using proteinase K or trypsin. For Western blot applications, compare reducing versus non-reducing conditions, as disulfide bonds in the EGF-like domains of MEGF10 may affect epitope accessibility. If studying MEGF10 in complexes, mild detergent conditions may preserve interactions but mask epitopes; in this case, test a panel of detergents with increasing stringency. Consider using alternative MEGF10 antibodies targeting different epitopes in parallel experiments. For proteins known to interact with MEGF10, such as ABCA1 , perform competition experiments with recombinant interacting proteins to determine if they affect antibody binding. Finally, complementary approaches such as epitope tagging of MEGF10 can provide alternative detection methods when antibody epitopes are inaccessible.

How can biotin-conjugated MEGF10 antibodies be utilized in super-resolution microscopy to study membrane protein organization?

For super-resolution microscopy applications, biotin-conjugated MEGF10 antibodies offer specific advantages when paired with appropriate detection methods. Begin by using streptavidin conjugated to photoswitchable fluorophores (such as Alexa Fluor 647) for STORM or PALM imaging, or with small quantum dots for single-molecule tracking. When preparing samples, optimize fixation to preserve membrane ultrastructure—mild fixation with 2% paraformaldehyde for 10-15 minutes is often suitable for membrane proteins like MEGF10 . For STED microscopy, use streptavidin conjugated to STED-compatible dyes such as STAR 635P. To study MEGF10 clustering at the membrane, implement quantitative cluster analysis using Ripley's K-function or DBSCAN algorithms on super-resolution datasets. For multi-color super-resolution imaging to study co-localization with ABCA1 or other interaction partners , carefully select fluorophore pairs with minimal cross-talk and implement sequential imaging strategies. Consider combining super-resolution with proximity ligation assays to visualize both nanoscale localization and direct interactions simultaneously. When analyzing super-resolution data, employ drift correction and fiducial markers to ensure accurate localization precision, particularly important for quantifying membrane protein organization patterns.

What approaches can combine ChIP-seq and MEGF10 antibody studies to investigate transcriptional regulation of MEGF10 expression?

To investigate transcriptional regulation of MEGF10, integrate ChIP-seq and antibody-based approaches through a systematic workflow. First, identify putative transcription factors regulating MEGF10 expression using bioinformatic analysis of the MEGF10 promoter region and enhancers. Perform ChIP-seq for these candidates in relevant cell types, focusing on factors related to phagocytosis or cell adhesion pathways given MEGF10's functions . Validate ChIP-seq findings using reporter assays with wild-type and mutated MEGF10 regulatory elements. To connect transcriptional regulation with protein expression, implement a time-course study where cells are exposed to stimuli predicted to alter MEGF10 expression, then analyze both transcription factor binding (via ChIP) and resulting MEGF10 protein levels (via Western blot with biotin-conjugated antibody). For mechanistic insights, conduct knockdown or overexpression of identified transcription factors and assess the impact on MEGF10 protein levels. Additionally, investigate epigenetic regulation by performing ChIP-seq for histone modifications and DNA methylation analysis across the MEGF10 locus. To establish physiological relevance, correlate chromatin states at the MEGF10 locus with protein expression levels across different tissues or disease states using tissue microarrays probed with the biotin-conjugated MEGF10 antibody.

How can researchers implement mass spectrometry-based approaches with biotin-conjugated MEGF10 antibodies to identify novel binding partners?

For identifying novel MEGF10 binding partners, implement an integrated immunoprecipitation-mass spectrometry (IP-MS) workflow. Leverage the biotin-conjugated MEGF10 antibody for efficient capture using streptavidin-coated magnetic beads, which provides clean isolation with minimal background. Perform crosslinking prior to cell lysis using membrane-permeable crosslinkers like DSP (dithiobis(succinimidyl propionate)) to capture transient interactions, particularly important for membrane proteins like MEGF10. Include appropriate controls: an IgG-biotin control immunoprecipitation and, ideally, a MEGF10-knockout cell line subjected to the same protocol. For analysis, implement both label-free quantification and SILAC approaches to distinguish true interactors from background contaminants. Given MEGF10's multiple reported functions in phagocytosis, cell adhesion, and proliferation , perform tissue-specific and context-dependent IP-MS experiments (e.g., during phagocytosis versus normal conditions). Validate top hits using reciprocal co-immunoprecipitation, proximity ligation assays, and functional studies. For challenging membrane protein interactions, consider implementing more specialized approaches such as BioID or APEX proximity labeling, where MEGF10 is fused to a biotin ligase or peroxidase to biotinylate proximal proteins, which can then be purified and identified by mass spectrometry.

What methodological considerations are important when designing MEGF10 knockout validation experiments using biotin-conjugated antibodies?

When validating MEGF10 knockout models, implement a comprehensive validation strategy using biotin-conjugated antibodies. Begin with genomic validation using PCR and sequencing to confirm the intended genetic modification. For protein-level validation, Western blot analysis using the biotin-conjugated MEGF10 antibody should demonstrate complete absence of the protein at the expected molecular weight of approximately 122 kDa . Include positive controls (wild-type samples) processed identically to confirm the antibody is working properly. For challenging cases where knockout verification is ambiguous, implement a panel approach using multiple MEGF10 antibodies targeting different epitopes. Perform immunofluorescence studies to confirm absence of MEGF10 localization at the cell membrane . For functional validation, assess phenotypes related to MEGF10's known functions, including phagocytosis efficiency, cell adhesion properties, and proliferation rates . Consider rescue experiments by re-expressing MEGF10 in knockout cells to confirm phenotype reversibility. When publishing knockout validation data, include full-length Western blot images with molecular weight markers to demonstrate absence of truncated products or cross-reactive bands. Additionally, perform RT-qPCR to confirm absence of transcript or to characterize any residual transcripts that might be produced from the modified locus.

How can researchers accurately interpret MEGF10 expression patterns across different tissues using biotin-conjugated antibodies?

To accurately interpret MEGF10 expression patterns across tissues, implement a comprehensive analytical framework. Begin by establishing a standardized protocol for tissue preparation, ensuring consistent fixation, antigen retrieval, and antibody incubation conditions across all samples. When using biotin-conjugated antibodies, be aware that certain tissues (particularly liver, kidney, and brain) contain high levels of endogenous biotin, which can lead to false-positive signals; implement appropriate blocking steps using avidin-biotin blocking kits. Conduct tissue microarray (TMA) studies with the biotin-conjugated MEGF10 antibody alongside markers for cell types known to express MEGF10, such as macrophages, to determine cell-type specific expression patterns. Quantify staining intensity using digital pathology tools with appropriate positive and negative controls on each slide. Validate immunohistochemistry findings with orthogonal methods such as in situ hybridization for MEGF10 mRNA and Western blotting of tissue lysates. When comparing pathological versus normal tissues, implement matched-pair analyses with blinded scoring. For developmental studies or disease progression analysis, clearly define and document changes in both expression levels and subcellular localization of MEGF10. Finally, correlate expression patterns with functional readouts related to MEGF10's known roles in phagocytosis and cell adhesion to establish biological relevance of the observed expression patterns.

What approaches help resolve contradictory findings between Western blot and immunofluorescence data for MEGF10 localization?

When facing contradictory results between Western blot and immunofluorescence data, implement a systematic troubleshooting approach. First, verify antibody specificity through knockout/knockdown controls in both applications. For membrane proteins like MEGF10 , subcellular fractionation followed by Western blotting can confirm membrane localization and potentially identify additional pools of the protein in other cellular compartments. Implement multiple fixation and permeabilization protocols for immunofluorescence, as different methods can dramatically affect epitope accessibility, particularly for membrane proteins. Consider that post-translational modifications might affect antibody recognition differently in native (immunofluorescence) versus denatured (Western blot) conditions. To address this, treat samples with phosphatases, glycosidases, or other enzymes that remove modifications before analysis. Employ super-resolution microscopy to more precisely define MEGF10 localization patterns that might be missed by conventional microscopy. For definitive localization studies, implement live-cell imaging using fluorescently tagged MEGF10 constructs and compare with antibody-based detection in fixed cells. Finally, consider that apparent contradictions might reflect biological reality—MEGF10 could shuttle between different cellular compartments depending on cell state, particularly given its roles in both membrane functions and intracellular vacuole formation . Document different experimental conditions thoroughly, as MEGF10 localization may be context-dependent.

How can researchers distinguish between specific and non-specific binding when using biotin-conjugated MEGF10 antibodies in complex tissue samples?

To distinguish specific from non-specific binding in complex tissues, implement a multi-layered validation approach. Begin with critical controls: include MEGF10 knockout tissue sections processed identically to experimental samples, and perform peptide competition assays where the antibody is pre-incubated with the immunizing peptide before tissue application. For biotin-conjugated antibodies specifically, include an endogenous biotin blocking step using avidin-biotin blocking kits, particularly important in biotin-rich tissues like liver, kidney, and brain. Implement dual-labeling approaches, combining the biotin-conjugated MEGF10 antibody with antibodies against known MEGF10-interacting proteins such as ABCA1 to confirm expected co-localization patterns. For challenging tissues, consider signal amplification methods with low background, such as tyramide signal amplification. Validate staining patterns across multiple biotin-conjugated MEGF10 antibodies targeting different epitopes, as consistent patterns across different antibodies strongly support specificity. When analyzing data, use quantitative approaches that account for background levels, implementing local background subtraction methods. Finally, correlate protein detection with mRNA expression using in situ hybridization on sequential sections to confirm that protein is detected in cells expressing the transcript. Document all optimization steps and controls in publications to support the validity of the observed staining patterns.

How can biotin-conjugated MEGF10 antibodies be applied in single-cell protein analysis techniques?

For applying biotin-conjugated MEGF10 antibodies in single-cell protein analysis, implement cutting-edge methodological approaches. For mass cytometry (CyTOF) applications, conjugate streptavidin with rare earth metals and use this to detect biotin-conjugated MEGF10 antibodies in a multi-parameter panel that includes markers for phagocytosis, cell adhesion, and proliferation pathways relevant to MEGF10 function . In microfluidic-based single-cell Western blotting, optimize protein separation parameters for high-molecular-weight proteins like MEGF10 (122 kDa) , using biotin-conjugated antibodies with fluorescent streptavidin for detection. For spatial proteomics applications such as Imaging Mass Cytometry or CODEX, incorporate the biotin-conjugated MEGF10 antibody into multiplexed panels to visualize MEGF10 expression in the spatial context of tissue architecture. When developing single-cell protein analysis workflows, validate that dissociation methods preserve MEGF10 epitopes, as aggressive dissociation can damage membrane proteins. For correlative single-cell transcriptomics and proteomics approaches (e.g., CITE-seq), evaluate whether cell surface MEGF10 can be detected using oligo-tagged streptavidin paired with the biotin-conjugated antibody. Finally, implement computational approaches that can integrate MEGF10 protein expression data with transcriptomic data at single-cell resolution to gain insights into post-transcriptional regulation of this important phagocytosis and cell adhesion mediator.

What considerations are important when using biotin-conjugated MEGF10 antibodies in neurodegenerative disease research models?

When applying biotin-conjugated MEGF10 antibodies in neurodegenerative disease research, several specialized considerations are critical. First, optimize detection protocols for neural tissues, where high lipid content and endogenous biotin can interfere with specific detection; implementing avidin-biotin blocking and testing multiple antigen retrieval methods is essential. Consider MEGF10's reported roles in phagocytosis in the context of microglial function, designing experiments to investigate whether MEGF10 participates in clearance of protein aggregates or damaged neurons in disease models. When working with aged brain tissues, address increased autofluorescence through appropriate quenching methods or spectral unmixing. For biotin-conjugated antibodies specifically, use streptavidin conjugated to far-red fluorophores to minimize overlap with tissue autofluorescence. Design co-localization studies examining MEGF10 in relation to disease-specific markers (e.g., amyloid plaques, neurofibrillary tangles) using multi-color immunofluorescence approaches. In mouse models of neurodegeneration, implement longitudinal studies examining MEGF10 expression and localization changes during disease progression, correlating with behavioral and neuropathological outcomes. Consider cell-type specific expression analyses focusing on microglia, astrocytes, and neurons to characterize potential shifts in MEGF10 expression patterns in disease states. Finally, validate findings from animal models in human post-mortem tissues, being mindful of potential epitope degradation issues associated with post-mortem delay.