GFRA2 Antibody

What is GFRA2 and what is its biological function?

GFRA2 (GDNF family receptor alpha 2) is a receptor for neurturin that belongs to the GDNFR family. It mediates the neurturin (NRTN)-induced autophosphorylation and activation of the RET receptor tyrosine kinase. GFRA2 is a potent survival factor for central dopaminergic neurons, motor neurons, and several other neuronal populations in both central and peripheral nervous systems. The protein plays crucial roles in neuronal development, maintenance, and signaling. Alternative names for GFRA2 include GDNFRB, RETL2, TRNR2, and NRTNR-ALPHA . The biological significance of GFRA2 extends to its involvement in neural development and potential implications in neurodegenerative diseases through its role in neuronal survival pathways .

What types of GFRA2 antibodies are available for research?

GFRA2 antibodies are predominantly available as rabbit polyclonal antibodies that target different epitopes of the protein. Most commercially available antibodies are unconjugated and suitable for various applications including Western blot (WB), immunohistochemistry (IHC), ELISA, and immunocytochemistry (ICC). These antibodies typically show reactivity with human, mouse, and rat samples, making them versatile tools for comparative research across species . Different antibodies target specific regions of GFRA2, such as amino acids 360-464, 377-391, or other segments, allowing researchers to select antibodies that recognize their region of interest depending on experimental requirements .

What are the expected molecular weights when detecting GFRA2?

The calculated molecular weight of GFRA2 is approximately 52 kDa (corresponding to 464 amino acids), but researchers should note that the observed molecular weight in experimental conditions frequently differs. Western blot analyses typically reveal bands at 34-40 kDa and/or 50-55 kDa . This discrepancy between calculated and observed molecular weights may be attributed to post-translational modifications, alternative splicing, or proteolytic processing. When validating antibody specificity, researchers should consider these multiple bands as potential indicators of different GFRA2 isoforms or modified forms rather than non-specific binding .

What are the optimal dilutions for GFRA2 antibodies in different applications?

The optimal dilution of GFRA2 antibodies varies depending on the specific application and the antibody source. For Western blot (WB) applications, recommended dilutions typically range from 1:200 to 1:2000, with many manufacturers suggesting a starting dilution of 1:500 . For immunohistochemistry (IHC), more concentrated preparations are often required, with recommended dilutions between 1:20 and 1:200 . It is advisable to perform titration experiments to determine the optimal concentration for each specific experimental system, as the optimal antibody concentration may vary based on tissue type, fixation method, and detection system. Sample-dependent optimization is crucial for obtaining reliable and reproducible results .

How should sample preparation be optimized for GFRA2 detection?

For effective GFRA2 detection, sample preparation protocols should be carefully optimized. For immunohistochemistry applications with GFRA2 antibodies, antigen retrieval using TE buffer at pH 9.0 is suggested, although citrate buffer at pH 6.0 can serve as an alternative . For Western blot applications, standard protocols for membrane protein extraction are generally suitable, considering GFRA2's cellular localization at the cell membrane as a GPI-anchored protein . When analyzing tissues, researchers should consider that GFRA2 expression has been positively detected in various samples including human liver tissue, mouse lung, rat brain, and cell lines such as HepG2, HeLa, LO2, and U-87MG . These positive samples can serve as appropriate positive controls for antibody validation experiments.

What positive controls should be used to validate GFRA2 antibody specificity?

To validate GFRA2 antibody specificity, several positive controls have been established. Cell lines with confirmed GFRA2 expression include HepG2, HeLa, LO2, and U-87MG . Tissue samples with known GFRA2 expression include human liver tissue, mouse lung, and rat brain . When designing validation experiments, researchers should include both positive controls (tissues/cells known to express GFRA2) and negative controls (tissues/cells with minimal or no GFRA2 expression) to confirm antibody specificity. Additionally, conducting knockdown or knockout experiments, where possible, provides the strongest evidence for antibody specificity by demonstrating reduced or absent signal in samples lacking the target protein .

Why do I observe multiple bands in Western blots using GFRA2 antibodies?

Multiple bands observed in Western blots using GFRA2 antibodies are a common occurrence that can be attributed to several biological and technical factors. The calculated molecular weight of GFRA2 is 52 kDa, but observed bands typically appear at 34-40 kDa and/or 50-55 kDa . These multiple bands may represent:

• Different isoforms resulting from alternative splicing of the GFRA2

• Post-translational modifications such as glycosylation, which is common for membrane receptors

• Proteolytic processing or degradation products

• Variable GPI-anchor attachment, considering GFRA2's localization as a GPI-anchored protein

To determine which bands represent specific GFRA2 detection, researchers should compare band patterns across different antibodies targeting different epitopes, perform blocking peptide experiments, or use genetic manipulation approaches (siRNA, CRISPR) to confirm band identity.

How can I distinguish between specific and non-specific staining in immunohistochemistry?

Distinguishing between specific and non-specific staining in immunohistochemistry (IHC) when using GFRA2 antibodies requires several validation steps. First, researchers should compare staining patterns with the known cellular localization of GFRA2 (cell membrane, GPI-anchored) . Specific staining should show membrane localization rather than diffuse cytoplasmic or nuclear patterns. Second, appropriate negative controls should be included, such as primary antibody omission, isotype controls, or pre-adsorption with immunizing peptides. Third, comparison of staining across multiple antibodies targeting different epitopes of GFRA2 can help confirm specificity. Finally, correlation with other detection methods (e.g., in situ hybridization for GFRA2 mRNA) can provide additional evidence for staining specificity. Optimizing antigen retrieval methods is also crucial, with suggested protocols using TE buffer at pH 9.0 or citrate buffer at pH 6.0 .

What are the common sources of variability in GFRA2 antibody performance across experiments?

Variability in GFRA2 antibody performance across experiments can stem from multiple sources that researchers should systematically address:

• Antibody lot-to-lot variation: Different production batches may show subtle differences in specificity and sensitivity

• Sample preparation variations: Differences in fixation methods, duration, and antigen retrieval protocols significantly impact epitope accessibility

• Cell/tissue type differences: GFRA2 may undergo different post-translational modifications or exist as different isoforms in various tissues

• Storage and handling conditions: Antibody activity can diminish with repeated freeze-thaw cycles or improper storage (recommended storage is at -20°C)

• Experimental conditions: Variations in blocking reagents, incubation times, and detection systems

To minimize variability, researchers should maintain detailed records of antibody lots, standardize protocols across experiments, and include consistent positive and negative controls in each experimental run.

How can GFRA2 antibodies be utilized in studies of neurodegenerative diseases?

GFRA2 antibodies offer valuable tools for investigating neurodegenerative disease mechanisms due to GFRA2's critical role in neuronal survival. These antibodies can be employed to:

• Examine changes in GFRA2 expression levels in patient samples or disease models using Western blotting and immunohistochemistry

• Investigate alterations in GFRA2 localization or trafficking in affected neurons through immunofluorescence studies

• Assess the integrity of neurturin-GFRA2-RET signaling pathways in disease states

• Identify potential therapeutic targets by monitoring GFRA2 expression in response to experimental treatments

The polyclonal antibodies available provide researchers with options to detect various epitopes and potentially different forms of GFRA2 in diseased tissues . By correlating GFRA2 expression patterns with disease progression or neuronal loss, researchers can gain insights into the protective or pathological roles of this receptor in conditions such as Parkinson's disease, where dopaminergic neurons (which express GFRA2) are primarily affected.

What approaches can be used to study GFRA2 interactions with the RET receptor and downstream signaling?

Studying GFRA2 interactions with the RET receptor and downstream signaling requires sophisticated approaches beyond basic antibody applications. Researchers can implement:

• Co-immunoprecipitation experiments using GFRA2 antibodies to pull down protein complexes and analyze RET co-precipitation

• Proximity ligation assays to visualize GFRA2-RET interactions in situ with spatial resolution

• Phospho-specific antibodies to monitor RET activation status following neurturin stimulation

• Immunofluorescence co-localization studies to track spatial relationships between GFRA2 and RET

• Functional assays combining GFRA2 antibodies with pharmacological inhibitors of downstream signaling pathways

When designing such experiments, researchers should consider that GFRA2 mediates the NRTN-induced autophosphorylation and activation of the RET receptor , and that appropriate controls for specificity are crucial when making claims about protein-protein interactions.

How can GFRA2 antibodies be combined with other techniques for comprehensive analysis of neuronal populations?

Integrating GFRA2 antibodies with complementary techniques enables comprehensive characterization of neuronal populations expressing this receptor. Advanced multi-modal approaches include:

• Coupling immunohistochemistry with laser capture microdissection to isolate GFRA2-positive neurons for subsequent molecular analysis

• Combining in situ hybridization for GFRA2 mRNA with immunostaining for protein to assess transcriptional and translational regulation

• Implementing flow cytometry with GFRA2 antibodies to quantify and isolate specific neuronal subpopulations

• Utilizing single-cell RNA sequencing data alongside immunohistochemistry to correlate GFRA2 protein expression with transcriptomic profiles

• Employing tissue clearing techniques with GFRA2 immunostaining for three-dimensional visualization of receptor distribution across intact neural circuits

These integrated approaches provide more comprehensive insights than any single method alone. When implementing multi-modal analyses, careful consideration must be given to compatibility between techniques, particularly regarding tissue preparation methods that satisfy the requirements of all analytical platforms being used.