GAS7 Antibody

What is GAS7 and why is it important in neuroscience research?

GAS7 (Growth Arrest Specific 7) is a protein that belongs to the F-BAR domain protein family, initially discovered in growth-arrested NIH3T3 fibroblasts . In humans, the canonical protein has a reported length of 476 amino acid residues and a mass of 54.4 kDa with cytoplasmic localization . GAS7 plays a crucial role in promoting maturation and morphological differentiation of cerebellar neurons . Its significance in neuroscience research stems from its involvement in actin polymerization and neurite outgrowth in various cultured cells, including embryonic primary cultured neurons . More recently, GAS7 has been identified as a regulator of mitochondrial dynamics within neurons of the central nervous system, with implications for neurodegenerative conditions like Parkinson's disease through its interaction with the PINK1 pathway .

What are the different isoforms of GAS7 and how are they detected?

GAS7 is expressed in three major isoforms: GAS7a, GAS7b, and GAS7c, which exhibit differential expression across various brain subregions . This diversity underscores the complexity of GAS7's function in neurodevelopment and repair. In Western blot analysis of mouse brain extracts, GAS7 antibodies typically detect three dominant protein bands at approximately 48 kDa, 55 kDa, and 63 kDa . When selecting antibodies for isoform detection, researchers should consider whether they need to detect all isoforms or specific ones, as antibodies raised against different epitopes may have varying detection capabilities. The subcellular expression pattern also varies, with GAS7 being detected prominently in the cytoplasm of both cell bodies and neurites of expressing cells .

What are the optimal protocols for Western blot detection of GAS7?

For reliable Western blot detection of GAS7:

• Sample preparation: Extract proteins from tissues or cells using standard lysis buffers containing protease inhibitors.

• Protein loading: Load 50μg of total protein per lane on SDS-PAGE (10-12% gel).

• Transfer: Transfer proteins to nitrocellulose membrane.

• Blocking: Block with 5% non-fat milk or BSA in TBST.

• Primary antibody: Dilute GAS7 antibody 1:500 to 1:1000 in blocking buffer and incubate overnight at 4°C .

• Secondary antibody: Incubate with appropriate HRP-conjugated secondary antibody.

• Detection: Visualize using an ECL detection system .

Expected results should include multiple bands corresponding to different GAS7 isoforms. When analyzing mouse brain tissue, three dominant bands of 48 kDa, 55 kDa, and 63 kDa are typically observed .

How can I optimize immunofluorescence staining for GAS7 in neuronal cells?

For optimal immunofluorescence staining of GAS7 in neuronal cells:

• Fixation: Fix cells with 4% paraformaldehyde for 15 minutes at room temperature.

• Permeabilization: Permeabilize with 0.1-0.3% Triton X-100 in PBS.

• Blocking: Block with appropriate serum in PBS containing 0.1% Triton X-100.

• Primary antibody: Dilute GAS7 antibody at 1:100 and incubate overnight at 4°C .

• Secondary antibody: Incubate with fluorophore-conjugated secondary antibody (e.g., Alexa fluor 488).

• Nuclear counterstain: Stain with DAPI for nucleus visualization .

For co-localization studies, GAS7 antibodies can be combined with markers like MAPII (for neurons), calbindin (for Purkinje neurons), or GFAP. Immunohistochemical analyses have shown high GAS7 expression in the cerebellum and hippocampus , with GAS7 detected prominently in the cell body and neurites of Purkinje neurons .

How can GAS7 antibodies be used to investigate mitochondrial dynamics?

Recent research has identified GAS7's role in mitochondrial dynamics and quality control through the PINK1 pathway . For such investigations:

• Mitochondrial co-localization:

  • Use double immunofluorescence with GAS7 antibody and mitochondrial markers

  • Apply confocal microscopy for detailed visualization

  • Compare wild-type versus GAS7-deficient samples

• Mitochondrial phenotype analysis:

  • Assess mitochondrial morphology (Gas7-deficient brain tissue shows elongated mitochondria with perinuclear clustering )

  • Evaluate mitochondrial fusion/fission protein levels

  • Examine PINK1-dependent phosphorylation of targets including Mfn-2 (S442), Parkin (S65), and ubiquitin (S65)

• Functional recovery assessment:

  • Test whether ectopic expression of GAS7 can restore normal mitochondrial morphology and distribution

  • Monitor PINK1 expression levels before and after GAS7 restoration

What are the best approaches for studying GAS7 in cerebellar neuron development?

For investigating GAS7's role in cerebellar neuron development:

• Primary culture system:

  • Establish primary cultures of embryonic cerebellar neurons

  • Use antibodies against cell-specific markers (MAPII, calbindin, GFAP) alongside GAS7 antibodies

  • Analyze temporal expression patterns during differentiation

• Cell-type specific expression:

  • GAS7 is expressed prominently in Purkinje neurons and to a lesser extent in granule neurons

  • GAS7 is detected in cells that produce MAPII and calbindin, but not in cells expressing GFAP

  • Use confocal microscopy to analyze cells doubly immunostained with relevant antibodies

• Functional studies:

  • Assess GAS7's role in promoting maturation and morphological differentiation

  • Monitor neurite outgrowth in relation to GAS7 expression

  • Consider knockdown/knockout approaches followed by rescue experiments

What controls should be included when using GAS7 antibodies in research?

Essential controls for GAS7 antibody experiments include:

• Negative controls:

  • Isotype controls (matching IgG) for immunoprecipitation and immunofluorescence

  • GAS7 knockout or knockdown samples

  • Secondary antibody-only controls for background assessment

• Positive controls:

  • Brain tissue samples (especially cerebellum and hippocampus)

  • Recombinant GAS7 protein as used in antibody generation

  • Cells with verified GAS7 expression (e.g., cerebellar neurons)

• Application-specific controls:

  • For Western blotting: Include molecular weight markers to verify isoform sizes

  • For immunofluorescence: Perform single-staining controls when conducting co-localization studies

  • For ELISA: Include standard curves with recombinant protein

Antibody validation should include testing against recombinant human GAS7 protein to ensure specificity before proceeding with experimental applications .

How can I resolve inconsistent results when using GAS7 antibodies?

When facing inconsistent results with GAS7 antibodies:

• Antibody selection considerations:

  • Verify antibody specificity (monoclonal antibodies like clone B-4 and AT4H8 provide higher specificity)

  • Consider epitope accessibility (antibodies targeting different regions may have varying efficacy)

  • Test multiple antibody clones if available

• Protocol optimization:

  • Adjust antibody concentration (recommended starting dilution 1:500 for Western blot)

  • Modify incubation times and temperatures

  • Try different blocking reagents to reduce background

• Sample preparation factors:

  • Ensure proper protein extraction and handling

  • Consider tissue-specific extraction methods

  • Use fresh samples when possible

• Cross-validation approaches:

  • Combine antibody detection with alternative methods (e.g., mRNA analysis)

  • Document all experimental conditions meticulously

  • Verify results across multiple experimental systems

Available GAS7 Antibody Products and Their Applications

GAS7 Expression Patterns in Different Brain Regions

Effects of GAS7 Deficiency on Mitochondrial Parameters