GFRA2 Antibody, Biotin conjugated

What is GFRA2 and what cellular functions does it mediate?

GFRA2 (GDNF family receptor alpha 2) belongs to the GDNFR family and functions as a receptor for neurturin. It mediates NRTN-induced autophosphorylation and activation of the RET receptor tyrosine kinase. GFRA2 serves as a potent survival factor for central dopaminergic neurons, motor neurons, and several other populations of neurons in both the central and peripheral nervous systems. The protein is involved in GDNF signaling, which is mediated by a complex of receptor tyrosine kinase RET and glial cell-derived neurotrophic factor family receptor-alpha (GFRA) . GFRA2 is also known as GDNFRB, RETL2, and TRNR2, reflecting its various roles in neuronal development and maintenance .

What are the molecular characteristics of GFRA2 antibodies with biotin conjugation?

GFRA2 antibodies with biotin conjugation typically consist of polyclonal antibodies raised in rabbits that recognize specific epitopes of the GFRA2 protein, with biotin molecules chemically attached to enhance detection capabilities. These antibodies have a calculated molecular weight targeting the full 464 amino acid GFRA2 protein (approximately 52 kDa), though observed molecular weights in experiments may range from 34-40 kDa and 50-55 kDa, likely due to post-translational modifications or alternative splicing . The biotin conjugation allows for strong and specific binding to streptavidin-based detection systems, creating a versatile tool for various experimental applications including ELISA and Western blotting, with recommended dilutions typically around 1:1000 for ELISA and 1:100-500 for Western blot applications .

What are the optimal dilution ratios for GFRA2 biotin-conjugated antibodies in different applications?

The optimal dilution ratios for GFRA2 biotin-conjugated antibodies vary significantly depending on the specific application, sample type, and detection method. For ELISA applications, a dilution ratio of 1:1000 is typically recommended to provide sufficient sensitivity while minimizing background . For Western blot applications, a more concentrated preparation ranging from 1:100 to 1:500 is generally advised to ensure adequate signal strength . When using these antibodies for immunohistochemistry applications, researchers should consider starting with dilutions between 1:20 and 1:200, with the understanding that optimal conditions may require titration based on the specific tissue being examined . For each new experimental setup, it is strongly recommended to perform a dilution series to determine the optimal antibody concentration that provides the best signal-to-noise ratio for your specific research context.

How should GFRA2 antibodies be stored and handled to maintain biotin conjugation efficacy?

GFRA2 biotin-conjugated antibodies require specific storage and handling conditions to maintain their efficacy and prevent degradation of both the antibody and biotin components. These antibodies should be stored at -20°C in a non-frost-free freezer, where they typically remain stable for one year after shipment . The storage buffer generally consists of PBS with 0.02% sodium azide and 50% glycerol at pH 7.3, which helps maintain protein stability during freeze-thaw cycles . While aliquoting is often recommended for antibodies to avoid repeated freeze-thaw cycles, some formulations of these antibodies (particularly those with glycerol) may not require aliquoting for -20°C storage . When working with the antibody, it should be thawed completely at room temperature or 4°C before use, gently mixed without vortexing, and kept on ice during experimental procedures to preserve the biotin conjugation and antibody binding capacity.

What controls should be included when using biotin-conjugated GFRA2 antibodies in various assays?

When using biotin-conjugated GFRA2 antibodies, a comprehensive set of controls is essential for experimental rigor and valid interpretation of results. First, a positive tissue control using samples known to express GFRA2 (such as HepG2 cells or human liver tissue) should be included to confirm antibody functionality . Equally important is a negative control utilizing tissues or cells that do not express GFRA2 to assess non-specific binding. An isotype control using rabbit IgG conjugated to biotin at the same concentration as the primary antibody helps distinguish between specific binding and Fc receptor interactions . For biotin-specific controls, researchers should include a streptavidin-only control (omitting primary antibody) to detect endogenous biotin in samples, and consider using avidin/biotin blocking kits particularly for tissue sections . Additionally, blocking peptide controls using the specific GFRA2 immunogen (such as the C-terminal epitope) can verify antibody specificity .

Why might multiple bands appear in Western blots when using GFRA2 biotin-conjugated antibodies?

Multiple bands in Western blots using GFRA2 biotin-conjugated antibodies can occur due to several biological and technical factors that researchers should systematically evaluate. The presence of bands at both 34-40 kDa and 50-55 kDa, as compared to the calculated molecular weight of 52 kDa, may indicate detection of different isoforms resulting from alternative splicing of GFRA2 mRNA, which has been documented in various experimental contexts . Post-translational modifications such as glycosylation, phosphorylation, or proteolytic processing can also alter the apparent molecular weight of GFRA2. Additionally, the observed bands might represent different maturation states of the protein, as GFRα2 undergoes processing including signal peptide cleavage and potential membrane association . Technical factors to consider include incomplete sample denaturation, excessive sample loading leading to non-specific binding, or inadequate blocking resulting in streptavidin binding to endogenous biotinylated proteins . To resolve these issues, researchers should optimize sample preparation conditions, include appropriate positive controls from tissues known to express GFRA2, and consider performing peptide competition assays to confirm band specificity.

How can background issues in immunohistochemistry with biotin-conjugated antibodies be minimized?

Background issues in immunohistochemistry using biotin-conjugated GFRA2 antibodies can be minimized through several targeted optimization strategies. The most significant source of background often stems from endogenous biotin in tissues, which can be effectively blocked by pre-incubating sections with avidin followed by biotin (avidin-biotin blocking kit) before applying the primary antibody . Optimizing fixation protocols is crucial, with recommendations suggesting antigen retrieval using TE buffer at pH 9.0 for GFRA2 detection, though citrate buffer at pH 6.0 may serve as an alternative . Implementing a more stringent blocking step using 5-10% normal serum from the same species as the secondary reagent, combined with 0.1-0.3% Triton X-100 for permeabilization, can significantly reduce non-specific binding . Dilution optimization is essential, with IHC applications typically requiring dilutions between 1:20-1:200, determined through systematic titration experiments . Additionally, reducing incubation time of the detection reagent, washing thoroughly between steps (at least 3 times for 5 minutes each), and using phosphate buffered saline with 0.05% Tween-20 (PBST) as a washing buffer can effectively minimize background while preserving specific GFRA2 signaling.

What accounts for discrepancies between calculated and observed molecular weights of GFRA2 in experimental data?

Discrepancies between the calculated molecular weight of GFRA2 (52 kDa based on its 464 amino acid sequence) and observed experimental weights (typically 34-40 kDa and 50-55 kDa) stem from multiple biological factors that influence protein migration in gel electrophoresis . Post-translational modifications, particularly glycosylation of the extracellular domain of GFRA2, can significantly increase the apparent molecular weight, explaining bands observed at 50-55 kDa . Conversely, proteolytic processing or alternative splicing of GFRA2 mRNA can generate shorter protein variants, accounting for the 34-40 kDa bands frequently detected in experimental settings . Research has demonstrated that GPS cell culture conditions can induce alternative splicing of GFRa2 mRNA, with distinct band shifts observed when comparing freshly isolated versus cultured cells . The hydrophobic nature of certain protein domains can also affect SDS binding and subsequent migration patterns. To accurately interpret these variations, researchers should compare results across multiple experimental systems, employ protein sequencing or mass spectrometry for definitive identification, and correlate observed bands with predicted splice variants documented in genomic databases .

How can GFRA2 biotin-conjugated antibodies be utilized in multiplex immunofluorescence studies?

GFRA2 biotin-conjugated antibodies offer significant advantages in multiplex immunofluorescence studies through versatile detection options and compatibility with complex staining protocols. For optimal implementation, researchers should pair these antibodies with streptavidin conjugated to fluorophores spectrally distinct from other channels in the experimental design, such as streptavidin-Alexa Fluor 647 or streptavidin-phycoerythrin . Sequential staining protocols are recommended when combining GFRA2 detection with other markers, applying the GFRA2 biotin-conjugated antibody first, followed by streptavidin-fluorophore, thorough washing, and then proceeding with additional antibody staining . When examining neuronal populations, GFRA2 can be effectively multiplexed with markers for RET receptor tyrosine kinase and neurturin to investigate the complete signaling complex . For pituitary stem cell research, combining GFRA2 detection with antibodies against Oct4, Prop1, and E-cadherin enables comprehensive characterization of the GPS (GRFa2/Prop1/Stem) cell niche . This approach has successfully demonstrated co-localization of these markers in spheroid structures derived from GFRA2-positive cell populations, revealing important insights about pituitary stem cell biology and differentiation potential .

What are the considerations when using GFRA2 biotin-conjugated antibodies in flow cytometry for cell sorting?

When employing GFRA2 biotin-conjugated antibodies for flow cytometry and cell sorting applications, researchers must address several critical technical considerations to ensure successful isolation of viable GFRA2-positive cell populations. First, cell preparation methodology significantly impacts results—collagenase digestion is strongly preferred over trypsin treatment since GFRα2 is an extracellular receptor highly sensitive to trypsin degradation, which can compromise antibody binding and subsequent sorting efficiency . Titration of the biotin-conjugated antibody is essential, typically starting with dilutions between 1:100-1:500, to determine optimal signal-to-noise ratio while minimizing non-specific binding . For detection, researchers should select streptavidin conjugates compatible with their cytometer configuration, ensuring the fluorophore brightness matches the expected expression level of GFRA2 in target populations . Critical validation steps include comparing sorted GFRα2+ and GFRα2- fractions via RT-PCR or immunofluorescence to confirm enrichment, with properly sorted fractions typically achieving 90-95% purity . Post-sorting viability assessment is particularly important, as studies have demonstrated that properly sorted GFRα2+ cells maintain proliferative capacity and can generate characteristic spheroid structures in appropriate culture conditions .

How can GFRA2 antibodies be used to study the GRFa2/Prop1/Stem (GPS) cell niche in pituitary research?

GFRA2 biotin-conjugated antibodies provide a powerful tool for studying the GRFa2/Prop1/Stem (GPS) cell niche in pituitary research through multiple complementary approaches. For identification and isolation of GPS cells, magnetic-activated cell sorting (MACS) or fluorescence-activated cell sorting (FACS) using biotin-conjugated GFRA2 antibodies allows researchers to obtain populations with approximately 90% purity, which can then be maintained in specialized spheroid medium (SpherM) to form characteristic spheroids with hollow cavities and motile cilia . Immunofluorescence characterization of these structures reveals co-expression of stemness markers including Oct4, Prop1, and E-cadherin, confirming their undifferentiated state . To assess GPS cell proliferation capacity, researchers can combine GFRA2 antibody staining with BrdU incorporation assays, which has demonstrated significant proliferative activity in purified GFRα2+ fractions compared to GFRα2- cells . For lineage tracing and differentiation studies, purified GPS cells can be cultured in differentiation medium (DifM), followed by immunostaining for both GFRA2 and differentiation markers such as Tubulin-beta III or growth hormone (GH) . RT-PCR analysis throughout the differentiation process reveals dynamic changes in gene expression patterns, including alternative splicing of GFRA2 mRNA during the transition from stemness to differentiated states .

What is the appropriate protocol for isolating GFRA2-positive cells using biotin-conjugated antibodies?

The isolation of GFRα2-positive cells using biotin-conjugated antibodies requires a carefully optimized protocol to ensure high cell viability and purity. Begin with fresh tissue preparation, preferentially using collagenase digestion rather than trypsin, as GFRα2 is an extracellular receptor sensitive to tryptic degradation . For magnetic-activated cell sorting (MACS), incubate the single-cell suspension with biotin-conjugated GFRA2 antibody (1:100-1:200 dilution) for 30 minutes at 4°C, followed by anti-biotin microbeads for 15 minutes . For fluorescence-activated cell sorting (FACS), use the biotin-conjugated primary antibody followed by streptavidin-fluorophore conjugate, maintaining cold conditions throughout to prevent receptor internalization . Post-isolation, verify purity through immunofluorescence assessment, with successful protocols typically achieving 90% GFRα2-positivity in the purified fraction and 95% negativity in the flow-through fraction . For subsequent culture and analysis, maintain isolated cells in specialized media such as SpherM to promote spheroid formation, with characteristic structures typically appearing after 5-7 days in culture . This protocol has been successfully implemented for both rat and mouse pituitary cells, demonstrating consistent results across rodent models .

What are the recommended antigen retrieval methods when using GFRA2 antibodies for tissue immunohistochemistry?

For optimal antigen retrieval when using GFRA2 antibodies in tissue immunohistochemistry, a systematic approach addressing both pH and buffer composition is essential. The primary recommended method utilizes TE buffer at pH 9.0, which has been documented to effectively unmask GFRA2 epitopes in various tissue types, including human liver samples . As an alternative approach, researchers may employ citrate buffer at pH 6.0, though this may yield variable results depending on tissue fixation conditions and specific epitope accessibility . The retrieval process should include heating the sections in the selected buffer using either a pressure cooker (3-5 minutes at full pressure) or microwave treatment (15-20 minutes at controlled temperature not exceeding 95°C) to optimize epitope exposure while preventing tissue damage . Following heat treatment, allowing sections to cool gradually in the retrieval solution for 20-30 minutes enhances antigen accessibility . For particularly challenging samples or when working with the C-terminal epitope of GFRA2, a combined approach of heat-mediated retrieval followed by limited proteolytic digestion (using proteinase K at 20 μg/ml for 5-10 minutes) may further improve antibody binding .

How should researchers design experiments to study GFRA2 alternative splicing using biotin-conjugated antibodies?

Designing experiments to study GFRA2 alternative splicing using biotin-conjugated antibodies requires a multifaceted approach combining protein and nucleic acid analysis techniques. Researchers should begin with Western blot analysis using biotin-conjugated GFRA2 antibodies targeting different epitopes (particularly C-terminal regions) to identify potential size variations indicative of splice variants, with expected bands ranging from 34-40 kDa and 50-55 kDa . Parallel RT-PCR analysis should be conducted using primers designed to span known or predicted splice junctions, which has previously revealed distinct band shifts in GFRα2 mRNA during cellular differentiation processes . For comprehensive characterization, isolate GFRα2-positive cells using biotin-conjugated antibodies via MACS or FACS, then maintain these populations in appropriate culture conditions (such as SpherM) to induce differentiation while collecting samples at defined timepoints for sequential analysis . This approach has successfully demonstrated changes in GFRA2 splicing patterns between freshly isolated cells and those cultured in spheroid-forming conditions . Additionally, researchers should perform immunofluorescence analysis of the differentiated cells using the biotin-conjugated antibodies to correlate protein expression patterns with the identified splice variants, providing insights into potential functional consequences of alternative splicing on GFRA2 signaling capabilities .

How should researchers quantify and compare GFRA2 expression levels across different experimental conditions?

Accurate quantification and comparison of GFRA2 expression levels across experimental conditions requires rigorous standardization and multiple complementary methodologies. For Western blot analysis using biotin-conjugated GFRA2 antibodies, researchers should implement densitometric quantification normalized to appropriate housekeeping proteins (such as β-actin or GAPDH), while accounting for all relevant GFRA2 bands (34-40 kDa and 50-55 kDa) that may represent different isoforms or post-translationally modified variants . When performing flow cytometry quantification, establish standardized gating strategies based on fluorescence minus one (FMO) controls and report data as median fluorescence intensity (MFI) ratios relative to isotype controls rather than simple percentage positive, providing more accurate assessment of expression level variations . For immunohistochemical evaluations, employ digital image analysis using standardized acquisition parameters and scoring systems that account for both staining intensity and percentage of positive cells, such as H-score or Allred methods . RT-qPCR analysis should complement protein-level measurements, with careful primer design to detect all relevant splice variants identified in previous studies . Most importantly, researchers should apply consistent methodologies across all experimental conditions being compared and include appropriate biological replicates (n≥3) to enable statistical validation of observed differences in GFRA2 expression levels.

What bioinformatic approaches can enhance interpretation of GFRA2 experimental data?

Bioinformatic approaches can significantly enhance interpretation of GFRA2 experimental data by providing contextual analysis and predictive insights across multiple dimensions of protein function. Researchers should integrate sequence analysis tools to identify conserved domains and potential post-translational modification sites that may explain the discrepancies between calculated (52 kDa) and observed molecular weights (34-40 kDa and 50-55 kDa) . Alternative splicing prediction algorithms combined with RNA-seq data analysis can help identify tissue-specific GFRA2 transcript variants, which have been documented during cellular differentiation processes . Protein-protein interaction network analysis should focus on known GFRA2 binding partners, particularly RET receptor tyrosine kinase and neurturin, to contextualize experimental findings within broader signaling pathways . For researchers investigating GPS cell populations, gene ontology enrichment analysis of differentially expressed genes between GFRα2+ and GFRα2- fractions can reveal functional categories associated with stemness and differentiation potential . Structural modeling approaches may predict how alternative splicing or post-translational modifications affect protein conformation and ligand binding capabilities. Additionally, integrating publicly available single-cell RNA-seq datasets can provide valuable insights into cell-type specific expression patterns of GFRA2 across different tissues and developmental stages, complementing experimental observations of GFRα2+ cell populations in specific niches .