GBA3 Antibody

What is GBA3 and why is it important in research?

GBA3, or cytosolic beta-glucosidase, is a protein predominantly expressed in liver tissue that efficiently hydrolyzes beta-D-glucoside and beta-D-galactoside compounds. Unlike its counterparts GBA1 (lysosomal) and GBA2 (microsomal), GBA3 functions in the cytosol and may be involved in detoxification of plant glycosides. Recent research indicates that GBA3 also possesses significant neutral glycosylceramidase activity, suggesting its involvement in a non-lysosomal catabolic pathway of glucosylceramide metabolism .

Notably, GBA3 shows significant homology (>40%) with Klotho protein, which is associated with aging processes . This connection makes GBA3 particularly interesting for researchers studying metabolic pathways and aging. The protein's broad substrate specificity toward various β-glycosides makes it an important target for studying cellular detoxification mechanisms .

What are the optimal applications for GBA3 antibodies in experimental design?

Based on available commercial antibodies, the most validated applications for GBA3 detection include:

For optimal experimental design, researchers should combine multiple detection methods. For instance, using both Western blot and immunocytochemistry provides complementary data on both protein expression levels and subcellular localization . When designing experiments, consider that GBA3 shows different molecular weights in different detection systems - approximately 53-57 kDa depending on the specific antibody and experimental conditions used .

How should GBA3 antibodies be stored and handled for maximum stability?

For maximum stability and reproducibility in experiments, GBA3 antibodies require specific storage and handling protocols:

• Storage temperature: Most GBA3 antibodies should be stored at -20°C to -70°C for long-term storage (up to 12 months from receipt) .

• Short-term storage: For antibodies in active use, store at 2-8°C under sterile conditions for up to 1 month after reconstitution .

• Aliquoting: To avoid repeated freeze-thaw cycles, prepare small working aliquots after reconstitution .

• Conjugated antibodies: For HRP-conjugated antibodies like NBP289924H, store at 4°C in the dark to prevent photobleaching of the conjugate .

• Buffer components: Most GBA3 antibodies are supplied in PBS with stabilizers like trehalose or glycerol, often with sodium azide as a preservative .

When preparing working dilutions, determine optimal concentration for each specific application through titration experiments. Remember that sodium azide, a common preservative in antibody preparations, inhibits HRP activity and should be avoided in peroxidase-conjugated detection systems .

What are the critical validation steps for confirming GBA3 antibody specificity?

Thorough validation is essential before using GBA3 antibodies in critical experiments:

• Multiple epitope recognition: Validate using antibodies targeting different epitopes of GBA3 (N-terminal vs. C-terminal). For instance, ABIN2787250 targets the N-terminal region, while other antibodies target internal or C-terminal domains .

• Protein band confirmation: GBA3 should appear at approximately 53-57 kDa on Western blots under reducing conditions, with some variability based on post-translational modifications and detection systems .

• Multi-tissue validation: Compare expression across tissues known to express GBA3 (primarily liver and kidney) versus low-expression tissues. Western blot data shows distinct bands in kidney cortex (AF5969) and both liver and kidney tissues (MAB5969) .

• Knockout/knockdown controls: When possible, validate using tissues or cells with GBA3 knockdown/knockout as negative controls.

• Enzymatic activity correlation: For functional studies, confirm that detected protein correlates with GBA3 enzymatic activity using substrates like pNP-β-Gal and pNP-β-Glc .

Cross-reactivity data indicates most antibodies react specifically with human GBA3, but some show cross-reactivity with other species including mouse, rat, dog, cow, guinea pig, pig, rabbit, and horse, with varying degrees of predicted reactivity .

How does GBA3 interact with NEU2, and what methodological approaches reveal this interaction?

Research has identified a significant interaction between GBA3 and NEU2 (a sialidase) that dramatically affects cytosolic sialyl free N-glycans (FNGs) metabolism. This interaction can be studied through several methodological approaches:

• Co-expression systems: The co-expression of NEU2 and GBA3 in MKN45 stomach cancer cells results in almost complete elimination of sialyl FNGs, suggesting a functional interaction .

• Biochemical interaction assays: Physical interaction between GBA3 and NEU2 can be confirmed through:

  • Co-immunoprecipitation assays with epitope-tagged proteins

  • Gel-filtration chromatography showing shifted elution profiles when proteins interact

• Protein stability studies: Cycloheximide-decay experiments demonstrate that GBA3 stabilizes NEU2 protein in cells. This can be quantified through time-course Western blotting after protein synthesis inhibition .

• In vitro enzyme activity assays: Mixing purified GBA3 and NEU2 proteins shows enhanced NEU2 activity compared to NEU2 alone, suggesting a functional interaction beyond physical binding .

The research indicates that while GBA3 shows broad substrate specificity toward various β-glycosides, it may not directly act on desialylated FNGs in vitro. Instead, its major effect appears to be stabilizing NEU2 activity, which enhances the degradation of sialyl FNGs .

What are the most effective troubleshooting strategies for GBA3 detection in challenging sample types?

When encountering difficulties detecting GBA3, particularly in samples with low expression or high background, consider these methodological approaches:

• Signal enhancement strategies:

  • For Western blotting: Use highly sensitive ECL substrates and concentrate protein lysates from low-expressing tissues

  • For immunostaining: Consider tyramide signal amplification systems when using HRP-conjugated antibodies like NBP289924H

• Background reduction:

  • Extensive blocking (5% BSA or milk for 1-2 hours)

  • Inclusion of 0.1-0.3% Triton X-100 in antibody diluents

  • For tissues with high endogenous biotin, use avidin/biotin blocking kits

• Sample preparation optimization:

  • For tissue samples: Optimize fixation - GBA3 detection works best in freshly isolated samples or those fixed with 4% paraformaldehyde

  • For cell fractionation: Use gentle detergents to preserve cytosolic fractions where GBA3 is primarily located

• Antibody selection based on application:

  • For detection of native protein: Polyclonal antibodies like AF5969 (sheep) or ABIN2787250 (rabbit) may provide better sensitivity

  • For specific applications requiring higher specificity: Monoclonal antibodies like MAB5969 (clone 728714)

• Species-specific considerations:

  • For human samples: Multiple validated antibodies are available

  • For non-human samples: Verify predicted reactivity and validate experimentally (e.g., predicted 93% reactivity for guinea pig and rabbit with ABIN2787250)

How can GBA3 antibodies be utilized in studies of glycoside metabolism and detoxification pathways?

GBA3's role in glycoside metabolism and potential detoxification functions can be investigated using several antibody-based approaches:

• Subcellular localization studies: Combine GBA3 antibodies with markers for different cellular compartments to confirm its cytosolic localization and potential redistribution under various conditions. Immunofluorescence approaches using antibodies like NBP289924H or Abbexa's GBA3 antibody are particularly useful for this purpose .

• Co-localization with metabolic enzymes: Use dual immunostaining to examine potential functional interactions between GBA3 and other glycoside-processing enzymes, particularly those involved in detoxification pathways.

• Expression analysis in response to xenobiotics: Monitor GBA3 expression changes after exposure to potential substrates or toxins using Western blot analysis with antibodies like AF5969 or MAB5969 .

• Tissue-specific expression profiling: Create comprehensive expression maps across tissues with varying detoxification requirements, capitalizing on the validated reactivity of antibodies in human liver and kidney tissues .

• Enzyme-antibody paired assays: Combine enzymatic activity measurements (using substrates like pNP-β-Gal and pNP-β-Glc) with quantitative immunoassays to correlate protein levels with functional activity in various physiological states .

These approaches can help elucidate GBA3's role in processing potentially harmful plant glycosides and contribute to understanding cytosolic detoxification mechanisms.

What is the significance of GBA3's homology with Klotho protein, and how can this be investigated?

The significant homology (>40%) between GBA3 and Klotho protein suggests potential shared functions or evolutionary relationships that may connect metabolism to aging processes . This relationship can be investigated through:

• Structural analysis approaches:

  • Epitope mapping using different GBA3 antibodies to identify structural domains shared with Klotho

  • Combining immunoprecipitation with mass spectrometry to identify shared interaction partners

• Functional comparative studies:

  • Parallel analysis of GBA3 and Klotho expression across tissues and aging models

  • Investigation of whether similar regulatory factors control both proteins

• Signal pathway analysis:

  • Using GBA3 antibodies to investigate whether GBA3, like Klotho, interfaces with specific signaling pathways related to aging

  • Co-immunoprecipitation studies to identify shared binding partners

• Aging model applications:

  • Quantitative analysis of GBA3 expression changes during aging using techniques like Western blotting with MAB5969 or AF5969

  • Immunohistochemical studies of age-related changes in GBA3 distribution within tissues

This research direction could provide new insights into the molecular mechanisms connecting metabolism to aging processes, potentially identifying GBA3 as a novel factor in age-related metabolic changes.

What methodological considerations are important when studying GBA3's role in non-lysosomal glucosylceramide metabolism?

GBA3's significant neutral glycosylceramidase activity suggests its involvement in a non-lysosomal catabolic pathway of glucosylceramide metabolism . When investigating this role, researchers should consider:

• Subcellular fractionation quality control:

  • Carefully separate cytosolic fractions to avoid contamination with lysosomal GBA1

  • Use antibodies specific to GBA3 (e.g., MAB5969) alongside markers for different cellular compartments

  • Verify fractionation quality through enzyme activity assays with compartment-specific substrates

• Simultaneous detection of multiple GBA isoforms:

  • Design multiplex immunofluorescence protocols using antibodies to different GBA isoforms

  • Employ multiple antibodies targeting different epitopes to ensure specificity

• Functional enzyme replacement studies:

  • Use siRNA knockdown of GBA3 followed by enzymatic activity assays

  • Monitor changes in glycosphingolipid levels after GBA3 manipulation

• Lipid metabolism analysis:

  • Combine GBA3 protein detection with lipidomics analyses

  • Correlate GBA3 levels with changes in ceramide and glucosylceramide levels across different physiological conditions

• Disease model applications:

  • Investigate GBA3 expression in conditions with dysregulated glycosphingolipid metabolism

  • Compare cytosolic versus lysosomal glucosylceramide processing in models of lipid storage disorders