AAGAB Antibody

What is AAGAB and why is it important in cellular research?

AAGAB encodes the alpha- and gamma-adaptin-binding protein p34, which interacts with the gamma-adaptin and alpha-adaptin subunits of complexes involved in clathrin-coated vesicle trafficking . It functions as an assembly chaperone governing the assembly of adaptor complexes 1 and 2 (AP1 and AP2) . AAGAB has been implicated in endocytic recycling of growth factor receptors such as EGFR, which can impact cell division and proliferation . Research on AAGAB is important for understanding fundamental cellular processes like membrane trafficking and has clinical relevance due to its connection to punctate palmoplantar keratoderma type 1 (PPKP1) and potential roles in cancer progression.

Which experimental applications are most suitable for AAGAB antibodies?

AAGAB antibodies have been validated for multiple research applications:

The selection of application should be based on research objectives, with WB being particularly effective for analyzing AAGAB expression levels and molecular weight confirmation .

What is the expected molecular weight for AAGAB detection in Western blotting?

While the predicted molecular weight of AAGAB is 35 kDa, researchers typically observe bands at 37-40 kDa due to post-translational modifications, particularly phosphorylation . When analyzing western blot results, it's important to note that truncated forms resulting from mutations may produce different banding patterns, which can be useful for mutation analysis studies . Using appropriate positive controls such as K-562 cells or mouse thymus tissue can help confirm correct band identification .

How should sample preparation be optimized for AAGAB antibody applications?

For optimal AAGAB detection, sample preparation methods should be tailored to the specific application:

• Western Blotting: Cell lysates should be prepared with buffers containing protease inhibitors to prevent degradation. Reducing conditions are recommended for most AAGAB antibodies .

• Immunohistochemistry: For tissue sections, antigen retrieval with TE buffer (pH 9.0) is recommended, though citrate buffer (pH 6.0) may be used as an alternative . Methanol-acetone fixation (1:1) for 5 minutes has been successfully used for cultured cells expressing AAGAB constructs .

• Cell-Based Assays: When studying AAGAB in cell culture models, consider using HaCaT keratinocytes, HeLa cells, or normal human keratinocytes, as these have been successfully used in published AAGAB studies .

What controls are essential when conducting AAGAB knockdown experiments?

When designing AAGAB knockdown studies using siRNA:

• Validated siRNA sequences: Use previously validated sequences such as "UGU AAG AGA GUG AGG AAU A" (AAGAB1239) and "GGA AAG UAC UGC AAA UAA A" (AAGAB2164) .

• Non-specific control siRNA: Include a scrambled sequence control (e.g., "UAG CGA CUA AAC ACA UCA AUU") to account for non-specific effects .

• Validation of knockdown: Confirm knockdown efficiency at both mRNA and protein levels using RT-PCR and western blotting.

• Functional readouts: Monitor the effects on AP complex stability, as AAGAB knockdown typically results in decreased levels of AP-4 ε and AP-4 β4 subunits and increased levels of the AP-4 cargo ATG9A .

• Time-course analysis: Assess knockdown effects at multiple timepoints (e.g., 72 and 96 hours post-transfection) to capture both early and late consequences .

How can researchers investigate AAGAB oligomerization states?

AAGAB has been shown to oligomerize in its resting state, with the C-terminal domain (CTD) mediating homodimerization by forming an antiparallel dimer . To study these oligomerization states:

• Analytical gel filtration: Use a Superdex 200 increase 10/300 GL column with HBS buffer (flow rate of 0.5 mL/min, injection volume of 0.5 mL) to analyze recombinant AAGAB proteins .

• SEC-MALS (Size Exclusion Chromatography coupled with Multi-Angle Light Scattering): This technique can determine the absolute molar mass and mass distribution of AAGAB proteins or complexes. Use a Superdex 200 Increase 10/300 column with DAWN HELEOS II and Optilab T-rEX detectors .

• Co-immunoprecipitation: To detect oligomerization in cells, co-express differently tagged versions of AAGAB (e.g., V5-tagged and 3xFLAG-tagged) and perform immunoprecipitation with antibodies against one tag (e.g., anti-FLAG) followed by western blotting for the other tag .

• Mutational analysis: Create mutations in the CTD (residues 258-301) to disrupt oligomerization and assess their effects on AAGAB function and AP complex stability .

What approaches can be used to study AAGAB interactions with adaptor protein complexes?

To investigate AAGAB's role as a chaperone for adaptor protein complexes:

• Co-expression and pull-down assays: Co-express GST-tagged AP subunits (e.g., GST-AP1γ or GST-AP2α) with His-SUMO-tagged AAGAB in E. coli, followed by either GST or nickel affinity pull-down .

• Binary complex purification: For AAGAB:AP2α binary complex purification, co-transform GST-AAGAB and His-SUMO-AP2α plasmids into BL21(DE3) E. coli cells, followed by sequential purification steps including nickel affinity chromatography, tag cleavage, and size exclusion chromatography .

• Cycloheximide chase experiments: To assess how AAGAB affects AP complex stability, perform CHX chase experiments in wild-type and AAGAB-knockout cells, with and without proteasomal inhibitors like MG132 .

• Mutagenesis studies: Create mutations in the AP1/2-binding region of AAGAB and assess their effects on AP complex association and stability .

How can AAGAB antibodies be used to study punctate palmoplantar keratoderma (PPKP1)?

PPKP1 is caused by heterozygous loss-of-function mutations in the AAGAB gene. To investigate PPKP1 pathomechanisms:

• Mutation detection: Perform PCR amplification of AAGAB exons followed by sequencing to identify mutations like c.370C>T (p.Arg124*) and c.481C>T (p.Arg161*) .

• Expression analysis: Use AAGAB antibodies for immunohistochemistry to compare protein expression and localization patterns between normal and PPKP1-affected skin samples. The granular cytoplasmic staining observed in control individuals is typically reduced in affected individuals .

• Functional consequences: Transfect cells with wild-type or mutant AAGAB constructs and assess differences in expression efficiency, localization, and effects on adaptor protein complexes .

• Haploinsufficiency modeling: Create heterozygous AAGAB knockout models using CRISPR-Cas9 targeting exons 1 or 2, and analyze the effects on AP complex stability and cargo trafficking .

What is the significance of AAGAB expression in cancer research?

AAGAB has potential diagnostic and prognostic implications in cancer research:

• Expression analysis: AAGAB expression is significantly upregulated in breast cancer compared to normal tissue, and higher expression correlates with worse prognosis .

• Immune infiltration correlation: AAGAB expression shows significant correlations with tumor purity (positive, Cor=0.326, p=7.9E-28) and immune cell infiltration, including positive correlations with CD8 T cells and macrophages, and negative correlations with CD4 T cells and dendritic cells .

• Prognostic marker: Kaplan-Meier survival analysis has shown that breast cancer patients with high AAGAB expression have worse prognosis than those with low expression (p=0.005) .

• Therapeutic considerations: AAGAB has been reported as a novel on-treatment biomarker that can improve prediction of response to neoadjuvant chemotherapy in breast cancer .

How can researchers troubleshoot non-specific binding in AAGAB antibody applications?

Non-specific binding is a common challenge when working with antibodies:

• Antibody selection: Choose antibodies that have been validated for specific applications and species of interest. For example, antibody ABIN1812721 has been validated for WB, IHC, and FACS in human samples .

• Blocking optimization: Adjust blocking conditions using 5% non-fat dry milk or BSA in TBST. The optimal blocking agent may vary depending on the application and specific antibody.

• Antibody dilution: Titrate antibody concentrations to determine the optimal dilution for each application. Recommended dilution ranges are 1:500-1:2000 for WB and 1:200-1:800 for IHC .

• Washing procedures: Implement rigorous washing steps (e.g., 3-5 washes of 5-10 minutes each with TBST) to reduce background signal.

• Secondary antibody controls: Include controls without primary antibody to identify non-specific binding from secondary antibodies.

What strategies can improve detection of low-abundance AAGAB in tissues?

For detecting low levels of AAGAB in tissue samples:

• Antigen retrieval optimization: Test both TE buffer (pH 9.0) and citrate buffer (pH 6.0) to determine optimal conditions for your specific tissue .

• Signal amplification: Consider using tyramide signal amplification (TSA) or polymer detection systems to enhance signal without increasing background.

• Tissue selection and processing: Use freshly fixed tissues when possible, as overfixation can mask epitopes. For AAGAB studies, thymus tissue has been reported as a reliable positive control .

• Concentration methods: For protein lysates with low AAGAB expression, consider immunoprecipitation prior to western blotting to concentrate the target protein.

• Alternative detection methods: In cases where traditional IHC yields insufficient results, consider RNAscope® or BaseScope™ assays to detect AAGAB mRNA as a proxy for protein expression.