AGTPBP1 Antibody

What is AGTPBP1 and what is its biological role?

AGTPBP1 (ATP/GTP Binding Protein 1) encodes cytosolic carboxypeptidase 1 (CCP1), which primarily functions in the deglutamylation of tubulin. This protein plays a critical role in regulating the assembly, transport, and signaling of microtubules, thereby maintaining the stability and function of cytoskeletal structures . The protein is widely expressed across multiple species, including humans, mice, rats, and other model organisms. At the molecular level, AGTPBP1 specifically cleaves poly-glutamic acids from the C terminus or side chains of α/β tubulins, contributing to post-translational modifications that regulate microtubule dynamics .

What antibody formats are available for AGTPBP1 research?

AGTPBP1 antibodies are available in multiple formats to accommodate diverse experimental needs:

• Host species: Primarily rabbit polyclonal and mouse monoclonal antibodies

• Clonality options: Both polyclonal and monoclonal (specific clones include 9A3, 2B9, 9B8, and 2N2)

• Binding specificity: Antibodies targeting different regions (N-terminal, internal region, C-terminal)

• Applications: Antibodies validated for Western blotting (WB), immunohistochemistry (IHC), immunohistochemistry-paraffin (IHC-P), ELISA, and flow cytometry (FACS)

When selecting an antibody, consider which specific epitope region you need to target and whether your experimental design requires broad epitope recognition (polyclonal) or precise epitope specificity (monoclonal).

What are the validated applications for AGTPBP1 antibodies?

AGTPBP1 antibodies have been validated for multiple research applications:

For optimal results, antibodies purified by peptide affinity chromatography (such as using SulfoLink™ Coupling Resin) generally provide better specificity and reduced background .

How should I optimize Western blotting protocols for AGTPBP1 detection?

When performing Western blotting to detect AGTPBP1, consider the following methodological recommendations:

• Sample preparation: AGTPBP1 is a relatively large protein (~140 kDa), requiring careful sample preparation to prevent degradation. Use freshly prepared RIPA buffer supplemented with protease inhibitors.

• Gel percentage: Use lower percentage gels (6-8% acrylamide) to effectively resolve the high molecular weight AGTPBP1 protein.

• Transfer conditions: For large proteins, extend transfer time or use wet transfer systems rather than semi-dry methods to ensure complete transfer.

• Blocking conditions: 5% non-fat dry milk in TBST has been shown to effectively reduce background without affecting specific AGTPBP1 antibody binding .

• Antibody dilution: Optimal dilution ranges for primary AGTPBP1 antibodies are typically between 1:500 to 1:2000, but should be empirically determined for each specific antibody .

• Controls: Include positive controls (cell lines known to express AGTPBP1, such as PANC-1 or HPAF-II pancreatic cancer cells) and negative controls (cells with siRNA-mediated AGTPBP1 knockdown) .

What are the recommended protocols for immunohistochemistry with AGTPBP1 antibodies?

For successful immunohistochemical detection of AGTPBP1 in tissue samples:

• Fixation: Standard formalin fixation (10% neutral-buffered formalin for 24-48 hours) provides consistent results for AGTPBP1 detection.

• Antigen retrieval: Heat-induced epitope retrieval using citrate buffer (pH 6.0) is recommended, as AGTPBP1 epitopes can be masked during fixation.

• Blocking: Use 5-10% normal serum from the same species as the secondary antibody to reduce non-specific binding.

• Antibody incubation: Primary AGTPBP1 antibody incubation at 4°C overnight generally provides optimal staining with minimal background.

• Detection systems: Both DAB-based chromogenic detection and fluorescence-based methods have been successfully used with AGTPBP1 antibodies .

• Counterstaining: Hematoxylin counterstaining provides good nuclear contrast when evaluating AGTPBP1 expression patterns.

• Controls: Include both positive control tissues (pancreatic cancer tissues have shown high AGTPBP1 expression) and negative controls (omission of primary antibody or tissues with confirmed low expression) .

How can AGTPBP1 antibodies be used to study its role in cancer progression?

AGTPBP1 has been implicated in cancer development, particularly in pancreatic cancer. Methodological approaches include:

• Expression analysis: Using AGTPBP1 antibodies for IHC to compare expression levels between tumor and adjacent non-tumor tissues. Research has shown AGTPBP1 is overexpressed in pancreatic cancer tissues compared to normal tissues .

• Correlation studies: Analyzing the relationship between AGTPBP1 expression levels and clinical parameters using semi-quantitative IHC scoring. Studies have found associations between AGTPBP1 expression and tumor location, particularly showing higher expression in tumors at the tail of the pancreas .

• Functional studies: Using AGTPBP1 antibodies to confirm knockdown efficiency in siRNA experiments aimed at studying the role of AGTPBP1 in cancer cell behaviors. Knockdown of AGTPBP1 has been shown to inhibit pancreatic cancer cell proliferation, migration, and invasion .

• Signaling pathway analysis: Western blotting with AGTPBP1 and downstream pathway antibodies has revealed that AGTPBP1 knockdown inhibits ERK1/2, P-ERK1/2, MYLK, and TUBB4B protein expression, suggesting involvement in the ERK signaling pathway .

What approaches are recommended for AGTPBP1 knockdown experiments?

For effective AGTPBP1 knockdown studies in cancer research:

• siRNA design: Multiple siRNA sequences targeting different regions of AGTPBP1 mRNA should be tested. In published research, the following siRNA sequences have demonstrated effective knockdown:

  • siAGTPBP1-1050: 5'-AGAUUGGCACCGCCAUGAUAA-3'

  • siAGTPBP1-2136: 5'-GCCUCCAUUCAAAGAGCCUAU-3'

  • siAGTPBP1-3463: 5'-UAUACCAUGGAGAGUACUUUA-3'

• Transfection optimization: Lipofectamine™ 2000 has been successfully used for transfection of AGTPBP1 siRNAs into pancreatic cancer cell lines with minimal cytotoxicity .

• Knockdown confirmation: Validate knockdown efficiency using:

  • RT-qPCR for mRNA level reduction

  • Western blotting with AGTPBP1 antibodies to confirm protein reduction

  • Immunofluorescence to visualize reduced expression at the cellular level

• Functional assays: After confirming knockdown, evaluate:

  • Cell proliferation using CCK-8 assays

  • Colony formation capability

  • Cell migration via wound healing assays

  • Invasion ability through transwell assays

How can I design experiments to study AGTPBP1's role in tubulin deglutamylation?

To investigate AGTPBP1's enzymatic function in tubulin deglutamylation:

• Co-immunoprecipitation: Use AGTPBP1 antibodies to pull down protein complexes, followed by Western blotting with anti-polyglutamylated tubulin antibodies to detect interaction.

• Enzyme activity assays: After immunoprecipitation with AGTPBP1 antibodies, the isolated protein can be used in in vitro deglutamylation assays with purified polyglutamylated tubulin as substrate.

• Cellular assays: Compare levels of polyglutamylated tubulin in control versus AGTPBP1-knockdown cells using:

  • Western blotting with specific antibodies against polyglutamylated tubulin

  • Immunofluorescence microscopy to visualize colocalization and changes in polyglutamylation patterns

• Rescue experiments: After AGTPBP1 knockdown, reintroduce wild-type or catalytically inactive AGTPBP1 to confirm the specificity of observed changes in tubulin polyglutamylation levels.

How can I validate the specificity of my AGTPBP1 antibody?

To ensure antibody specificity:

• Positive and negative controls: Use cell lines with confirmed high AGTPBP1 expression (e.g., PANC-1 cells) and compare with normal cells with lower expression (e.g., HPDE6-C7) .

• Peptide competition assay: Pre-incubate the AGTPBP1 antibody with its immunizing peptide before application to samples. Specific antibody binding should be blocked by the peptide.

• siRNA knockdown validation: Compare antibody signal in control versus AGTPBP1-knockdown samples. A specific antibody will show reduced or absent signal in knockdown samples .

• Cross-species reactivity: Verify reactivity across species if working with multiple model organisms. Many AGTPBP1 antibodies have been validated for reactivity with human, mouse, and rat proteins, with predicted reactivity in additional species .

What are common challenges when working with AGTPBP1 antibodies?

Researchers commonly encounter these challenges when working with AGTPBP1 antibodies:

• Background signal in IHC: This can be reduced by:

  • Optimizing blocking conditions (use 5-10% serum from the same species as the secondary antibody)

  • Extending washing steps between antibody incubations

  • Using antibodies purified by peptide affinity chromatography

• Weak signal in Western blotting: Improve signal by:

  • Increasing protein loading amount (start with 50-75 μg of total protein)

  • Using extended exposure times with sensitive detection systems

  • Optimizing transfer conditions for high molecular weight proteins

  • Using fresh lysates, as AGTPBP1 may be susceptible to degradation during storage

• Antibody cross-reactivity: When investigating AGTPBP1 in complex samples, verify antibody specificity using knockout/knockdown controls to ensure observed signals are specific to AGTPBP1.

How can AGTPBP1 antibodies help investigate its role in drug resistance?

Recent studies suggest connections between tubulin glutamylation levels and cancer drug resistance. AGTPBP1 antibodies can be instrumental in investigating:

• Expression correlation: Compare AGTPBP1 expression levels in drug-sensitive versus drug-resistant cancer cell populations using Western blotting or IHC.

• Drug response assays: After AGTPBP1 knockdown or overexpression, evaluate changes in sensitivity to microtubule-targeting drugs (e.g., paclitaxel, estramustine, nocodazole) .

• Combination therapy studies: Use AGTPBP1 antibodies to monitor changes in expression and activity when testing combinations of microtubule-targeting drugs with inhibitors of related pathways.

• Mechanistic investigation: Examine how AGTPBP1-mediated changes in tubulin post-translational modifications affect drug binding sites and microtubule dynamics in the context of drug resistance .

How are AGTPBP1 antibodies used in angiogenesis research?

AGTPBP1 has been implicated in tumor angiogenesis, with knockdown showing reduced blood vessel formation in xenograft models. Methodological approaches include:

• Tumor xenograft models: AGTPBP1 knockdown in cancer cells (e.g., PANC-1) before subcutaneous injection into nude mice results in tumors with significantly reduced blood vessel formation .

• Angiogenesis markers: Use AGTPBP1 antibodies alongside angiogenesis markers such as CD31 in IHC to evaluate correlation between AGTPBP1 expression and vascular density in tumors .

• In vitro angiogenesis assays: Investigate effects of conditioned media from AGTPBP1-knockdown cancer cells on endothelial cell tube formation, migration, and proliferation.

• Signaling pathway analysis: Combine AGTPBP1 antibodies with antibodies against angiogenic factors (VEGF, bFGF) and related signaling molecules to determine mechanistic connections.