GLUD2 Antibody

What is GLUD2 and where is it primarily expressed in human tissues?

GLUD2 (Glutamate Dehydrogenase 2) is a protein belonging to the glutamate receptor delta family. In the central nervous system, GLUD2 exhibits a distinctive expression pattern, being highly enriched in the molecular layer and Purkinje cells of the cerebellum . This specific distribution pattern is crucial for researchers to understand when designing experiments involving cerebellar tissue. Western blot analyses have confirmed GLUD2 expression in human brain cortex tissue, human testis tissue, and in mouse and rat brain cortex tissues . The protein typically appears at approximately 58 kDa on Western blots under reducing conditions .

Beyond neural tissues, immunohistochemistry studies have demonstrated GLUD2 localization in astrocytes within rat brain tissue . Additional detection has been reported in human prostate cancer tissue via immunohistochemistry , suggesting potential relevance in oncology research contexts. This diverse tissue distribution makes GLUD2 an important target for multiple research areas, from neuroscience to cancer biology.

How should researchers assess the specificity of commercial GLUD2 antibodies?

Commercial GLUD2 antibodies demonstrate varying degrees of specificity that must be rigorously validated. A comprehensive specificity assessment should include multiple complementary approaches. First, researchers should verify immunoreactivity patterns in tissues with known GLUD2 expression, particularly in cerebellum where GLUD2 is highly enriched in the molecular layer and Purkinje cells . Any staining pattern that does not match this distinctive distribution should be scrutinized.

For biochemical validation, Western blot analysis should demonstrate a primary band at approximately 58 kDa, which is the expected molecular weight of GLUD2 . Additionally, cell-based assays using GLUD2-transfected cells provide a powerful system for specificity validation. Expression verification can be performed with commercial antibodies against known epitopes, such as "a rabbit polyclonal antibody against an intracellular epitope corresponding to the center region of the Human GRID2" or antibodies against specific amino acid residues of GLUD2 .

Cross-reactivity with GLUD1 should be carefully evaluated, as many commercial antibodies are labeled as "GLUD1/GLUD2" antibodies, suggesting they recognize epitopes common to both proteins . When experimental goals require distinguishing between these related proteins, researchers should consider antibodies targeting regions with known sequence differences between GLUD1 and GLUD2, and validate specificity through additional methods such as recombinant protein controls or knockdown models.

What are the optimal sample preparation techniques for GLUD2 antibody detection?

Sample preparation protocols significantly impact GLUD2 antibody detection and vary by application. For Western blot analysis, lysates should be prepared under reducing conditions using appropriate buffer systems (e.g., Immunoblot Buffer Group 1) . PVDF membranes have yielded good results for GLUD2 detection, and proper sample loading concentration is crucial (approximately 0.5 mg/mL for brain tissue) .

For immunohistochemistry applications, perfusion-fixed frozen sections have been effectively used for GLUD2 detection in brain tissue . Antigen retrieval is particularly critical, with recommended protocols including TE buffer (pH 9.0) as the primary recommendation and citrate buffer (pH 6.0) as an alternative option . The importance of antigen retrieval cannot be overstated, as it significantly impacts epitope accessibility and staining quality. Counterstaining with hematoxylin provides good contrast for DAB-detected GLUD2 .

For cell-based assays, both permeabilized and non-permeabilized (live) cell protocols have been utilized, depending on whether intracellular or extracellular epitopes are being targeted . When investigating autoantibodies, researchers should compare standard two-step detection methods with multi-step approaches, though it's important to note that the three-step method "did not improve antibody detection and showed more frequent nonspecific reactivity that was not immunoabsorbed with GLUD2" .

What are the recommended applications and dilutions for GLUD2 antibodies?

GLUD2 antibodies have been validated for multiple research applications, each with specific technical parameters for optimal results. For Western blot applications, dilutions ranging from 1:5000 to 1:50000 have been successfully employed . This wide dilution range suggests that researchers should optimize concentrations for their specific experimental conditions. When performing immunohistochemistry, dilutions between 1:20 to 1:200 are recommended , with attention to appropriate antigen retrieval methods as discussed previously.

For immunofluorescence and immunocytochemistry applications, recommended dilutions typically range from 1:50 to 1:500 . These techniques have been validated in several cell lines, including HepG2 cells. When utilizing GLUD2 antibodies in cell-based assays, particularly for autoantibody detection research, both standard two-step methods and three-step detection protocols have been described .

Additionally, GLUD2 antibodies have been validated for use in Simple Western™ automated capillary-based immunoassays, particularly for detection in human brain tissue . Importantly, sample-dependent optimization is often necessary to achieve optimal results across these applications, and preliminary titration experiments are strongly recommended when establishing new protocols or working with unfamiliar sample types.

How do developmental factors influence GLUD2 detection in tissue samples?

Developmental factors can significantly impact GLUD2 detection in tissue samples, particularly in the context of cerebellar tissue analysis. One study emphasized that "selection of patients' sera and cerebellar tissue from very young rats (equivalent to 18–24 human months) were critical for antigen precipitation" . This suggests that the developmental stage of tissue samples may influence GLUD2 expression levels, post-translational modifications, or epitope accessibility.

When working with developmental tissues, researchers should consider age-matched controls and standardized tissue collection protocols. The expression pattern of GLUD2 in the cerebellum, primarily in the molecular layer and Purkinje cells , may show developmental variations that impact antibody binding. Additionally, fixation parameters may need adjustment for developmental tissues, as protein crosslinking can differ between immature and mature tissues.

For studies comparing GLUD2 expression across developmental stages, consistency in tissue processing, fixation methods, and detection protocols is essential to ensure comparable results. When investigating potential autoantibodies against GLUD2, the age of the tissue used for immunoprecipitation or immunohistochemistry may significantly influence results, as demonstrated by the emphasis on young cerebellar tissue in previous studies .

How do you troubleshoot inconsistent GLUD2 antibody staining patterns in cerebellar tissue?

Inconsistent GLUD2 antibody staining patterns in cerebellar tissue can arise from multiple methodological factors. First, researchers should verify whether the observed pattern matches the expected localization of GLUD2, which is "highly enriched in the molecular layer and Purkinje cells of cerebellum" . Any deviation from this characteristic pattern warrants careful investigation of technical parameters.

Tissue preparation and fixation protocols significantly impact GLUD2 detection. Optimization strategies should include comparing perfusion-fixed versus post-fixed tissue preparations, testing multiple fixative formulations and durations, and adjusting section thickness for frozen sections. Developmental considerations are particularly important, as one study emphasized that "cerebellar tissue from very young rats (equivalent to 18–24 human months) were critical for antigen precipitation" .

Antigen retrieval optimization is crucial for consistent results. Researchers should systematically compare the recommended protocols using TE buffer (pH 9.0) versus citrate buffer (pH 6.0) , varying retrieval duration and temperature, and evaluating microwave versus water bath methods. Detection system parameters also require optimization, including direct versus indirect detection methods and different visualization systems.

To address potential cross-reactivity issues, incorporate appropriate blocking steps, conduct pre-absorption studies with the target antigen, and consider immunodepletion approaches to confirm specificity. When using multiple antibodies, compare staining patterns from antibodies targeting different epitopes of GLUD2, such as those against "the amino acid residues 852–931 of mouse GluD2" versus those against "the amino acid residues 206–218 of rat GluD2" .

What are the contradictory findings regarding GLUD2 antibodies in opsoclonus-myoclonus syndrome?

The literature contains significant contradictions regarding the role of GLUD2 (GluD2) antibodies in opsoclonus-myoclonus syndrome (OMS). These contradictions center on whether GluD2 antibodies represent a reliable biomarker for this neurological condition.

A study by Berridge et al. reported that 14 of 16 children with OMS (87.5%) had GluD2 antibodies, suggesting these antibodies could serve as biomarkers . This study utilized mass spectrometry and bioinformatic techniques with age-equivalent cerebellar tissue to identify GluD2 as an autoantigen . The authors emphasized that selecting young rat cerebellar tissue was critical for antigen precipitation and observed intense reactivity with the granular cell layer and deep cerebellar nuclei of adult rat brain sections .

In direct contradiction, a study by Sabater et al. found no GluD2 antibodies in 203 patients with OMS (45 children, 158 adults) . These researchers noted that the expected pattern of GluD2 expression is in the molecular layer and Purkinje cells of cerebellum, not the granular layer and deep nuclei as reported by Berridge et al. . Their methodology employed three different detection techniques: rat brain immunohistochemistry, a live cell-based assay with standard secondary antibody, and a cell-based assay with secondary and tertiary antibodies . They concluded that "GluD2-ab should not be considered diagnostic biomarkers of OMS" .

Several methodological differences may explain these contradictory findings, including tissue age, detection methods, sample size, and interpretation of staining patterns. The Sabater study used a much larger cohort (203 OMS patients versus 16 in Berridge's study) and emphasized that their three-step method "did not improve antibody detection and showed more frequent nonspecific reactivity that was not immunoabsorbed with GluD2" .

How should researchers design cell-based assays for detecting GLUD2 antibodies?

When designing cell-based assays (CBAs) for GLUD2 antibody detection, researchers should consider several critical technical parameters. For the expression system, previous studies have successfully used "complementary DNA encoding the full-length human GluD2 mature polypeptide (GenBank ID NM_001510; Asp24-Ile1007) [...] cloned into the pHLsec vector" . Expression verification is essential and can be facilitated by including epitope tags; one study used "GluD2 fused to an extracellular HA tag" to confirm surface expression.

The choice between live and fixed cell assays depends on the experimental objective. For detecting autoantibodies binding to extracellular domains, live (non-permeabilized) cell assays are preferred, as demonstrated by verification with "a commercial antibody against the intracellular C-terminus of GluD2 (figure 2A; permeabilized CBA) and with a commercial antibody to the extracellular HA tag on the surface of live (nonpermeabilized) GluD2-transfected cells" . For intracellular epitopes, fixed and permeabilized cells are necessary.

Rigorous controls are essential, including vector-only transfected cells as negative controls, commercial anti-GLUD2 antibodies as positive controls, and for patient sample testing, known positive and negative control samples. Immunoabsorption tests can confirm specificity, as demonstrated when "Immunoabsorption of the samples with HEK293T cells expressing GluD2 did not abrogate the equivocal staining, indicating that it was not antigen-specific" .

What strategies are effective for optimizing immunoprecipitation protocols with GLUD2 antibodies?

Optimizing immunoprecipitation (IP) protocols for GLUD2 antibodies requires careful consideration of multiple parameters. Tissue selection is critical, with previous research emphasizing age-appropriate samples: "mass spectrometry and bioinformatic techniques using age-equivalent cerebellar tissue were used to identify GluD2 as an autoantigen" . For cerebellar tissue specifically, one study highlighted the importance of using "cerebellar tissue from very young rats (equivalent to 18–24 human months)" .

Antibody selection for immunoprecipitation should be based on validated IP performance. For studies of autoantibodies, purified IgG from patient samples has been successfully employed: "Precipitated OMAS IgG-antigen complexes were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis analysis and compared to a control sera" . Lysis and buffer conditions must be optimized to effectively solubilize GLUD2 while preserving antibody-binding epitopes, with particular attention to buffer composition based on whether targeting intracellular or extracellular epitopes.

For verification of precipitated proteins, multiple approaches are recommended. Western blot analysis using commercial anti-GLUD2 antibodies can confirm identity, while mass spectrometry provides unbiased identification: "The excised bands identified GluD2 in the patient but not control samples" . Stringent filtering of mass spectrometry data is crucial: "The eluates typically contained approximately 18,000 peptides matching to several hundred proteins were isolated from gel bands. To identify targets of potentially pathogenic autoantibodies, stringent filters were applied to filter out proteins present in the controls, and from the unique ones, to identify cerebellar-specific membrane proteins with an extracellular domain. This reduced the putative target pool to 12 proteins" .

Comprehensive controls must include isotype-matched control antibodies, control samples processed in parallel, and for autoantibody studies, comparison to "control sera" . Validation of IP results with orthogonal methods is recommended, as exemplified by the statement: "This identification of GluD2 as an autoantigen was confirmed using a CBA" .

How should researchers assess cross-reactivity between GLUD2 antibodies and other glutamate receptors?

Assessing cross-reactivity between GLUD2 antibodies and other glutamate receptors requires a multi-faceted approach. Bioinformatic analysis of sequence homology between GLUD2 and other glutamate receptor family members provides a foundation, with particular focus on regions targeted by specific antibodies, such as "the amino acid residues 852–931 of mouse GluD2" or "the amino acid residues 206–218 of rat GluD2" .

Recombinant protein panel testing offers direct assessment of cross-reactivity. By expressing a panel of glutamate receptor proteins and testing GLUD2 antibodies against this panel via Western blot or ELISA, researchers can identify potential cross-reactive targets. Cell-based assay approaches provide another valuable method, in which HEK293T cells are transfected separately with each glutamate receptor family member and GLUD2 antibody binding is assessed using protocols similar to those described: "HEK293T cells were transfected with complementary DNA encoding GluD2 fused to an extracellular HA tag" .

Competitive binding assays, where GLUD2 antibodies are pre-incubated with purified GLUD2, can determine whether binding to other glutamate receptors is blocked (suggesting shared epitopes) or unaffected (indicating distinct binding sites). Immunoabsorption studies provide further evidence of specificity, as demonstrated when "Immunoabsorption of the samples with HEK293T cells expressing GluD2 did not abrogate the equivocal staining, indicating that it was not antigen-specific" .

Tissue immunostaining pattern analysis is particularly valuable, comparing observed patterns with known expression patterns of different glutamate receptors. For GLUD2, cerebellar staining should show "intense reactivity with Purkinje cells and the molecular layer of cerebellum (where GluD2 is highly enriched)" . Knockout/knockdown validation provides definitive evidence, as persistent binding in knockout samples would indicate cross-reactivity.

What experimental controls are essential when using GLUD2 antibodies for Western blotting?

Comprehensive control strategies for Western blot analysis using GLUD2 antibodies are essential for reliable results. Positive control samples should include tissues with established GLUD2 expression, such as human brain (cortex) tissue, human testis tissue, and mouse and rat brain cortex tissues , as well as cell lines with confirmed GLUD2 expression, including A431 cells, HepG2 cells, HeLa cells, and U-251 cells .

Negative control samples are equally important, including tissues with minimal GLUD2 expression and samples processed with secondary antibody only. Molecular weight markers covering the expected size range are crucial, as GLUD2 typically appears at approximately 58 kDa under reducing conditions . In immunoprecipitation studies, a band of approximately 100-110 kDa has been observed , suggesting possible dimers or complexes.

For antibody specificity verification, parallel blots with multiple antibodies targeting different GLUD2 epitopes provide valuable cross-validation. Technical controls should include processing samples under both reducing and non-reducing conditions to identify potential structural impacts on epitope recognition. The appropriate membrane type and buffer system are also important: "PVDF membrane was probed with 0.2 μg/mL of Mouse Anti-Human/Mouse/Rat GLUD1/GLUD2 Monoclonal Antibody" and "This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 1" .

Loading and transfer controls ensure consistent sample processing, including housekeeping protein detection and Ponceau S staining of membranes. Detection system controls should include secondary antibody-only lanes to assess non-specific binding. Finally, antibody validation controls involve testing different antibody concentrations to determine optimal working dilution (recommended range: 1:5000-1:50000) and assessing batch-to-batch consistency with reference samples.

What are the implications of detecting GLUD2 autoantibodies in patient samples?

Methodological considerations are paramount when testing patient samples. The approach used by Sabater et al. was particularly comprehensive, examining "serum of 45 children with OMS (10 [22%] with neuroblastoma), 158 adults with OMS (53 [34%] with tumors), and 172 controls including 134 patients with several types of neurologic disorders, 18 with neuroblastoma without OMS, and 20 healthy participants" . Multiple detection methods should be employed, as demonstrated by their use of three different techniques: rat brain immunohistochemistry, a live cell-based assay using a standard secondary antibody, and a cell-based assay with secondary and tertiary antibodies .

The potential for non-specific reactivity presents a significant challenge, as illustrated by the observation that "The 3-step method (as reported in reference 2) was also negative in all cases but showed more frequent equivocal reactivity" . This highlights the importance of stringent specificity controls when assessing autoantibodies in patient samples.