GABARAPL1 Antibody

What is GABARAPL1 and why is it important in research?

GABARAPL1 (GABA(A) receptor-associated protein like 1) belongs to the GABARAP family of proteins and plays crucial roles in autophagy and receptor trafficking. It was initially identified as an estrogen-regulated gene and has been implicated in multiple cellular processes. The protein functions as a ubiquitin-like modifier that increases cell-surface expression of kappa-type opioid receptors by facilitating their anterograde intracellular trafficking . GABARAPL1 is also involved in autophagosome formation and maturation .

The protein has multiple synonyms including ATG8, GEC1, APG8L, ATG8L, and APG8-LIKE, which sometimes causes confusion in the literature . Due to its involvement in autophagy, GABARAPL1 has become a target of interest in research on neurodegenerative diseases, cancer, and other conditions where autophagy dysregulation occurs .

How do I choose between polyclonal and monoclonal GABARAPL1 antibodies for my experiment?

The selection between polyclonal and monoclonal antibodies depends on your specific experimental requirements:

Polyclonal Antibodies:

• Recognize multiple epitopes on GABARAPL1

• Often provide higher sensitivity, particularly useful for low abundance targets

• Available options include rabbit polyclonal antibodies like 18721-1-AP and CAB7790

• Better for applications where signal amplification is important

• May show batch-to-batch variability

Monoclonal Antibodies:

• Recognize a single epitope

• Provide high specificity with minimal cross-reactivity

• Available as mouse monoclonal (66458-1-Ig) or rabbit monoclonal (D5R9Y)

• GABARAPL1 (D5R9Y) XP® specifically does not cross-react with other GABARAP family members

• Better for quantitative applications requiring reproducibility

For initial characterization studies, a polyclonal antibody may be preferable, while for long-term studies requiring consistency, a monoclonal antibody would be more suitable .

What is the expected molecular weight for GABARAPL1 detection in Western blot applications?

While the calculated molecular weight of GABARAPL1 is 14 kDa, the observed molecular weight in Western blot experiments typically ranges between 16-18 kDa . This discrepancy may be attributed to post-translational modifications or structural properties affecting protein migration in SDS-PAGE gels.

Different antibodies report slightly different observed weights:

• 18721-1-AP detects GABARAPL1 at approximately 17 kDa

• 66458-1-Ig detects the protein at 16-18 kDa

• D5R9Y XP® antibody detects bands at both 14 kDa and 16 kDa

When optimizing Western blot protocols, it's important to account for this variation and include appropriate positive controls such as mouse brain tissue, which shows high expression of GABARAPL1 .

What are the optimal dilutions for different applications of GABARAPL1 antibodies?

Optimal antibody dilutions vary by application type and specific antibody. Below is a compilation of recommended dilutions from multiple sources:

It's generally recommended to perform a dilution series during initial optimization for each new experimental system. Begin with the manufacturer's recommended range and adjust based on signal intensity and background levels .

Which tissue or cell samples are best to use as positive controls for GABARAPL1 detection?

Based on validation data from multiple antibody manufacturers, the following samples serve as reliable positive controls for GABARAPL1 detection:

Tissue samples:

• Mouse brain tissue (consistently shows strong expression)

• Rat brain tissue

• Human heart tissue

• Human spleen tissue

• Mouse kidney tissue

• Mouse liver tissue

Cell lines:

• HepG2 cells (validated for both Western blot and immunofluorescence)

When establishing a new assay, including one of these validated positive controls alongside your experimental samples is crucial for confirming antibody performance and optimizing detection conditions .

How can I validate the specificity of my GABARAPL1 antibody?

Multiple approaches can be employed to validate GABARAPL1 antibody specificity:

• Knockdown/Knockout Validation:

  • Utilize siRNA knockdown or CRISPR/Cas9 knockout of GABARAPL1

  • Compare signal between wild-type and KD/KO samples

  • A specific antibody will show significantly reduced signal in KD/KO samples

• Peptide Competition Assay:

  • Pre-incubate antibody with immunizing peptide before application

  • Specific binding will be blocked by the peptide, resulting in signal reduction

• Cross-Reactivity Testing:

  • Test against recombinant GABARAPL1 and related family members (GABARAP, GABARAPL2)

  • Some antibodies like D5R9Y XP® are validated not to cross-react with other GABARAP family members

• Multiple Antibody Comparison:

  • Use antibodies targeting different epitopes of GABARAPL1

  • Consistent results across antibodies increase confidence in specificity

• Mass Spectrometry Verification:

  • For advanced validation, immunoprecipitate GABARAPL1 and confirm identity by mass spectrometry

What are common pitfalls when using GABARAPL1 antibodies in immunohistochemistry (IHC)?

Several challenges may arise when using GABARAPL1 antibodies for IHC:

• Antigen Retrieval Optimization:

  • GABARAPL1 detection often requires specific antigen retrieval conditions

  • For 18721-1-AP antibody, TE buffer pH 9.0 is suggested for optimal results

  • Alternatively, citrate buffer pH 6.0 may be used, but comparative testing is recommended

• Endogenous Autophagy Fluctuations:

  • GABARAPL1 expression levels vary depending on autophagic state

  • Consider standardizing sample collection timing or using autophagy modulators in control experiments

• Fixation Sensitivity:

  • Overfixation can mask epitopes

  • Standardize fixation protocols (duration, temperature, fixative concentration)

• Background Reduction:

  • Use appropriate blocking reagents (5% normal serum from the same species as the secondary antibody)

  • Include proper negative controls (omit primary antibody or use isotype control)

• Dilution Optimization:

  • Start with the recommended 1:50-1:500 dilution range and optimize

  • Test multiple dilutions on the same tissue type to determine optimal signal-to-noise ratio

How can I improve signal detection in Western blot applications for GABARAPL1?

To enhance GABARAPL1 detection in Western blots:

• Sample Preparation:

  • Include protease inhibitors in lysis buffer to prevent degradation

  • Consider using directly denatured samples rather than reducing agents separately

  • For tissues rich in lipids (like brain), optimize extraction buffers

• Loading Amount Optimization:

  • GABARAPL1 may require higher loading amounts (30-50 μg) of total protein for clear detection

  • Brain tissue samples typically require less protein due to higher expression levels

• Transfer Conditions:

  • Use PVDF membranes rather than nitrocellulose for better protein retention

  • Optimize transfer time for small proteins (14-18 kDa) - typically 60-90 minutes is sufficient

  • Consider semi-dry transfer systems for improved efficiency with small proteins

• Blocking Optimization:

  • Test both BSA and non-fat dry milk as blocking agents

  • 5% BSA often yields better results for phospho-specific antibodies

• Antibody Incubation:

  • Extend primary antibody incubation to overnight at 4°C

  • Consider using signal enhancers compatible with your detection system

• Detection Systems:

  • ECL-Plus or other enhanced chemiluminescent substrates improve sensitivity

  • For very low abundance, consider fluorescent secondary antibodies and imaging

What buffer and storage conditions are recommended to maintain GABARAPL1 antibody stability?

Most GABARAPL1 antibodies are formulated and stored under similar conditions for optimal stability:

• Storage Buffer Composition:

  • PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 is commonly used

  • Some formulations include 0.1% BSA for additional stability

  • Carrier-free formulations may use phosphate buffers (Na₂HPO₄, KCl, KH₂PO₄, NaCl) at pH 7.8

• Temperature Requirements:

  • Store at -20°C for long-term stability

  • Most formulations are stable for one year after shipment when properly stored

• Aliquoting Guidelines:

  • For antibodies in 50% glycerol, aliquoting is generally unnecessary for -20°C storage

  • For formulations without glycerol, aliquot to avoid freeze-thaw cycles

  • If slight precipitate forms, gentle vortexing can dissolve it without affecting performance

• Working Solution Handling:

  • Diluted antibody working solutions should be prepared fresh

  • If storage is necessary, keep at 4°C with preservatives for no more than 1-2 weeks

How can GABARAPL1 antibodies be used to monitor autophagy flux in experimental systems?

GABARAPL1 serves as a valuable marker for monitoring autophagy, particularly in later stages of autophagosome maturation. To effectively study autophagy flux using GABARAPL1 antibodies:

• Dual Detection Strategy:

  • Monitor both non-lipidated (cytosolic) and lipidated (membrane-bound) forms of GABARAPL1

  • The lipidated form typically appears as a faster-migrating band in Western blots

  • Use gels with higher acrylamide percentage (15-16%) for better separation of these forms

• Autophagy Flux Analysis:

  • Combine GABARAPL1 detection with autophagic flux inhibitors such as bafilomycin A1 or chloroquine

  • Increased accumulation of lipidated GABARAPL1 after treatment indicates active autophagy

• Comparative Analysis with Other Autophagy Markers:

  • While LC3s are involved in phagophore membrane elongation, GABARAPL1 is crucial for later stages of autophagosome maturation

  • Combine GABARAPL1 detection with LC3 and p62/SQSTM1 for comprehensive autophagy assessment

• Immunofluorescence Applications:

  • Use IF to visualize autophagosomal structures

  • GABARAPL1 puncta formation correlates with autophagosome formation

  • Co-localization with lysosomes (LAMP1/2) indicates autophagosome-lysosome fusion

• Quantitative Assessment:

  • Use ratiometric analysis of lipidated vs. non-lipidated forms

  • Quantify number and size of GABARAPL1-positive puncta in IF experiments

What is the significance of GABARAPL1 in xenotransplantation research and how are antibodies used in this context?

GABARAPL1 has emerged as a significant factor in xenotransplantation research, particularly in studies addressing rejection mechanisms:

How can I distinguish between GABARAPL1 and other closely related GABARAP family members in my experiments?

Distinguishing between closely related GABARAP family members requires careful experimental design:

• Antibody Selection:

  • Use antibodies specifically validated for GABARAPL1 selectivity

  • The D5R9Y XP® Rabbit mAb has been validated not to cross-react with other GABARAP family members

  • For other antibodies, perform validation using recombinant proteins of each family member

• Sequence Comparison Approach:

  • GABARAPL1 shares high sequence homology with other family members

  • Target antibodies to the most divergent regions, typically the N-terminal region

• Molecular Weight Differentiation:

  • GABARAPL1: Observed at 16-18 kDa

  • GABARAP: Typically observed at 14-16 kDa

  • GABARAPL2/GATE-16: Observed at approximately 16 kDa

  • Use high-percentage gels (15-18%) for better separation

• Expression Pattern Analysis:

  • GABARAPL1 shows particularly high expression in brain, heart, and liver tissues

  • Different family members show distinct tissue expression patterns

  • Use known differential expression patterns to help validate specificity

• Genetic Approaches:

  • In cell culture models, use siRNA specific to GABARAPL1 to confirm antibody specificity

  • For advanced studies, generate knockout cell lines for each family member

How should I interpret variations in GABARAPL1 detection between different tissue samples?

When analyzing GABARAPL1 expression across different tissues:

• Normal Tissue Expression Patterns:

  • GABARAPL1 is expressed at very high levels in brain, heart, peripheral blood leukocytes, liver, kidney, and placenta

  • Brain tissue consistently shows strong expression and serves as a reliable positive control

  • Expression can vary significantly between organs and should be normalized accordingly

• Developmental Considerations:

  • GABARAPL1 expression patterns may change during development

  • Age-matched samples should be used for comparative studies

• Cellular Localization Variations:

  • GABARAPL1 can be found in multiple cellular compartments including cytoplasm, cytoplasmic vesicles, autophagosome, cytoplasmic vesicle membrane, cytoskeleton, endoplasmic reticulum, and Golgi apparatus

  • Different tissues may show different predominant localizations

  • Use subcellular fractionation or co-localization studies to clarify distribution

• Autophagic State Influence:

  • Autophagy levels vary by tissue type and physiological/pathological conditions

  • Nutritional status, stress conditions, and disease states affect GABARAPL1 expression

  • Control for these variables when making cross-tissue comparisons

• Technical Considerations:

  • Different tissues may require unique extraction methods for optimal protein recovery

  • Loading controls should be carefully selected based on their stability across the tissues being compared

What experimental design is recommended for studying GABARAPL1's role in the endoplasmic reticulum stress response?

GABARAPL1 participates in the remodeling of endoplasmic reticulum (ER) subdomains into autophagosomes during nutrient stress . A comprehensive experimental design to study this role should include:

• ER Stress Induction Models:

  • Pharmacological inducers: tunicamycin (N-glycosylation inhibitor), thapsigargin (SERCA inhibitor), brefeldin A (ER-Golgi transport inhibitor)

  • Physiological stressors: glucose deprivation, amino acid starvation, hypoxia

  • Include time-course experiments (6h, 12h, 24h) to capture dynamic responses

• GABARAPL1 Expression and Localization Analysis:

  • Western blot to monitor protein levels under different ER stress conditions

  • Immunofluorescence to visualize co-localization with ER markers (KDEL, calnexin) and autophagy markers (LC3)

  • Live-cell imaging with fluorescently tagged GABARAPL1 to track dynamic changes

• Interaction with TEX264:

  • Co-immunoprecipitation to detect GABARAPL1-TEX264 interaction during ER stress

  • Proximity ligation assay to visualize interaction in situ

  • TEX264 knockdown to assess dependency of GABARAPL1 recruitment to ER subdomains

• Functional Assays:

  • Autophagic flux measurement using tandem fluorescent LC3 (mRFP-GFP-LC3) in combination with GABARAPL1 detection

  • ER-phagy assays measuring turnover of ER resident proteins (e.g., FAM134B, RTN3) in the presence/absence of GABARAPL1

  • Cell viability assays under ER stress conditions with GABARAPL1 modulation

• Genetic Manipulation Approaches:

  • CRISPR/Cas9 knockout of GABARAPL1

  • Rescue experiments with wild-type vs. mutant GABARAPL1 lacking specific interaction domains

  • Point mutations in the LC3-interacting region (LIR) of TEX264 to disrupt GABARAPL1 binding

What considerations are important when designing experiments to study GABARAPL1 post-translational modifications?

GABARAPL1 undergoes several post-translational modifications that affect its function. When designing experiments to study these modifications:

• Lipidation Detection:

  • GABARAPL1, like other ATG8 family proteins, undergoes lipidation (conjugation to phosphatidylethanolamine)

  • Use 15-16% gels for clear separation of lipidated and non-lipidated forms

  • Include phosphatase inhibitors in lysis buffers to preserve modifications

  • Consider urea-containing gels for enhanced separation of lipidated forms

• Phosphorylation Analysis:

  • Include phosphatase inhibitors in all extraction buffers

  • Use Phos-tag™ gels for enhanced separation of phosphorylated forms

  • Combine with phospho-specific antibodies or mass spectrometry for site identification

  • Consider in vitro kinase assays to identify responsible kinases

• Ubiquitination Studies:

  • Include deubiquitinase inhibitors (e.g., N-ethylmaleimide) in lysis buffers

  • Perform immunoprecipitation under denaturing conditions to disrupt non-covalent interactions

  • Use tandem ubiquitin binding entities (TUBEs) to enrich ubiquitinated proteins

• Mass Spectrometry Approaches:

  • Immunoprecipitate GABARAPL1 under conditions that preserve modifications

  • Use both bottom-up (peptide) and top-down (intact protein) MS approaches

  • Consider enrichment strategies for specific modifications before MS analysis

• Site-Directed Mutagenesis:

  • Generate mutants at predicted modification sites

  • Compare functional consequences of these mutations in cellular assays

  • Create non-modifiable and phosphomimetic mutations to study functional impacts