CAPZA1 Antibody

What is CAPZA1 and what role does it play in cytoskeletal regulation?

CAPZA1 is a member of the F-actin capping protein alpha subunit family. It functions by binding to the barbed (fast-growing) ends of actin filaments in a Ca²⁺-independent manner, effectively preventing further polymerization and depolymerization . Unlike other capping proteins such as gelsolin and severin, CAPZA1 does not sever actin filaments. It forms a heterodimer with CAPZB, stabilizing the dynactin complex structure and activating the molecular motor dynein for ultra-processive transport along microtubules . This regulatory function is essential for maintaining proper actin cytoskeleton architecture and dynamics in various cellular processes.

When performing Western blotting for CAPZA1 detection, consider these optimization strategies:

• Sample preparation: CAPZA1 has a molecular weight of approximately 33 kDa . Use appropriate lysis buffers containing protease inhibitors to prevent degradation.

• Antibody dilution: Different antibodies require different dilutions. For example:

  • Proteintech antibody (11806-1-AP): 1:500-1:1000

  • Boster Bio antibody (A07665): 1:200-1:1000

  • Cell Signaling antibody (#62114): 1:1000

• Blocking conditions: Use 5% non-fat dry milk or BSA in TBST for optimal results.

• Incubation times: Primary antibody incubation overnight at 4°C generally yields better results than shorter incubations at room temperature.

• Controls: Include positive controls such as human brain tissue, which has been confirmed to express CAPZA1 .

How can I validate the specificity of my CAPZA1 antibody?

Ensuring antibody specificity is critical for reliable results. Implement these validation methods:

• Knockout/knockdown controls: Use CAPZA1 siRNA (e.g., sequences 5′-CUG UGA AGA UAG AAG GAU A-3′ or 5′-GGA ACA AGA UAC UCA GCU A-3′) to generate negative controls .

• Cross-reactivity testing: Verify that your antibody does not cross-react with related proteins such as CAPZA2. Some antibodies, like Cell Signaling's E3U1G Rabbit mAb, are specifically tested not to cross-react with CAPZA2 .

• Immunoprecipitation followed by mass spectrometry: This can confirm that the antibody is capturing the intended target.

• Multiple antibody approach: Use antibodies targeting different epitopes of CAPZA1 to confirm results.

How can CAPZA1 antibodies be used to investigate its role in cancer progression?

CAPZA1 has emerging significance in cancer research, with different roles depending on cancer type:

• Gastric cancer: CAPZA1 overexpression is associated with better prognosis in gastric cancer patients . Researchers can use IHC with CAPZA1 antibodies to assess expression levels in patient samples. Studies suggest using a scoring system based on percentage of positively stained cells: 0 (0%), 1+ (1–24%), 2+ (25–49%), 3+ (50–74%), and 4+ (75–100%) .

• Hepatocellular carcinoma (HCC): CAPZA1 inhibits epithelial-mesenchymal transition (EMT), migration, and invasion in HCC . Experimental approaches include:

  • Western blotting and qPCR to detect CAPZA1 and EMT markers (E-cadherin, N-cadherin, Vimentin)

  • Transwell migration and invasion assays to assess cell motility

  • Animal models with orthotopic transplantation of CAPZA1-manipulated HCC cells monitored by MRI

When investigating correlations between CAPZA1 expression and EMT markers, researchers should simultaneously examine EMT transcription factors such as Snail1 and ZEB1, which have been shown to negatively correlate with CAPZA1 expression levels .

What methodological approaches can be used to study CAPZA1's interaction with the actin cytoskeleton?

To investigate CAPZA1's interactions with actin filaments:

• Co-immunoprecipitation (Co-IP): Use CAPZA1 antibodies for IP experiments to pull down associated proteins. For example, Proteintech's antibody (11806-1-AP) has been validated for IP at 0.5-4.0 μg for 1.0-3.0 mg of total protein lysate .

• Fluorescence microscopy with dual-labeling: Apply CAPZA1 antibodies in conjunction with actin staining (phalloidin) to visualize co-localization at actin filament barbed ends.

• Proximity ligation assay (PLA): This technique can detect protein-protein interactions between CAPZA1 and potential binding partners in situ with spatial resolution.

• Super-resolution microscopy: Techniques such as STORM or PALM can provide nanoscale resolution of CAPZA1 localization relative to actin filaments.

How can I establish effective CAPZA1 overexpression or knockdown systems for functional studies?

For manipulating CAPZA1 expression levels:

• Overexpression systems:

  • Clone human CAPZA1 cDNA using PCR with primers such as 5′-AGCTAAGCTTCCACCATGGCCGACTTCGATGAT-3′ and 5′-AATTGAATTCTTAAGCATTCTGCATTTCTTT-3′

  • Insert into appropriate expression vectors (e.g., pCMV-Tag2B/G418)

  • Transfect target cells (e.g., MKN-45 cells) using reagents like TerboFect™

  • Select stable transfectants using neomycin

  • Confirm overexpression by Western blot analysis

• Knockdown approaches:

  • Use validated siRNA sequences targeting CAPZA1:

    • 5′-CUG UGA AGA UAG AAG GAU A-3′ (siCAPZA1-A)

    • 5′-GGA ACA AGA UAC UCA GCU A-3′ (siCAPZA1-B)

  • Transfect using reagents like siLentiFect™

  • Harvest cells after 24-48 hours

  • Confirm knockdown efficiency by Western blot analysis

• CRISPR/Cas9 genome editing: For complete knockout studies, design guide RNAs targeting CAPZA1 exons and select clones with confirmed mutations.

What are the critical considerations when using CAPZA1 antibodies for immunohistochemistry in tissue samples?

For optimal IHC results with CAPZA1 antibodies:

• Antigen retrieval: Use either TE buffer (pH 9.0) or citrate buffer (pH 6.0) based on the antibody specifications. For example, Proteintech's antibody (11806-1-AP) recommends TE buffer pH 9.0 for optimal results .

• Antibody dilution: Typical dilutions range from 1:20 to 1:200 for IHC applications, but this should be optimized for each tissue type and fixation method .

• Blocking conditions: Blocking in 10% goat serum at room temperature for 1 hour before antibody incubation helps reduce background staining .

• Incubation conditions: Overnight incubation at 4°C with the primary antibody typically yields the best results .

• Detection systems: Use appropriate detection systems compatible with the host species of the primary antibody, such as anti-mouse/rabbit universal immunohistochemical detection kits .

How can CAPZA1 antibodies be used to investigate its role in epithelial-mesenchymal transition (EMT)?

To study CAPZA1's role in EMT regulation:

• Expression correlation analysis: Examine correlations between CAPZA1 expression and EMT markers (E-cadherin, N-cadherin, Vimentin) through Western blotting and immunofluorescence.

• Transcription factor analysis: Investigate how CAPZA1 affects EMT-related transcription factors like Snail1 and ZEB1, which have been shown to be negatively correlated with CAPZA1 expression levels .

• Functional migration/invasion assays: Use transwell migration and invasion assays to determine how CAPZA1 expression levels affect cell motility and invasiveness .

• In vivo metastasis models: Employ orthotopic transplantation of CAPZA1-manipulated cancer cells in animal models and monitor metastasis using imaging techniques like MRI .

• Actin cytoskeleton analysis: Since CAPZA1 regulates actin dynamics, examine how altered CAPZA1 expression affects stress fiber formation and cell morphology changes associated with EMT.

What are common troubleshooting strategies for non-specific binding when using CAPZA1 antibodies?

When encountering non-specific binding:

• Antibody dilution optimization: If background is high, try increasing the dilution. For example, for Western blot applications, try using higher dilutions (1:1000) of antibodies that are recommended at 1:500-1:1000 .

• Blocking optimization: Test different blocking agents (BSA vs. non-fat dry milk) and increase blocking time.

• Washing steps: Increase the number and duration of washing steps with TBST or PBS-T.

• Alternative antibodies: Consider switching to monoclonal antibodies like Cell Signaling's E3U1G Rabbit mAb (#62114) which may offer higher specificity than polyclonal antibodies .

• Pre-adsorption: If available, use blocking peptides corresponding to the immunogen used to generate the antibody to confirm specificity.

How can I design experiments to investigate CAPZA1's interaction with the dynactin complex?

To study CAPZA1's role in the dynactin complex:

• Co-immunoprecipitation: Pull down CAPZA1 and blot for dynactin components or vice versa.

• Proximity ligation assay: Detect in situ interaction between CAPZA1 and dynactin components.

• Live-cell imaging: Use fluorescently tagged CAPZA1 and dynactin components to track their co-localization and dynamics during vesicular transport.

• CAPZA1 mutant expression: Express mutant forms of CAPZA1 that disrupt its interaction with dynactin and observe effects on microtubule-based transport.

• Super-resolution microscopy: Apply techniques like STED or STORM to visualize the nanoscale organization of CAPZA1 within the dynactin complex.

What are effective strategies for studying post-translational modifications of CAPZA1?

To investigate post-translational modifications:

• Phospho-specific antibodies: Though not mentioned in the search results, these can be developed to study specific phosphorylation sites on CAPZA1.

• Mass spectrometry: Immunoprecipitate CAPZA1 and analyze by mass spectrometry to identify phosphorylation, acetylation, or ubiquitination sites.

• 2D gel electrophoresis: This can separate different post-translationally modified forms of CAPZA1 based on charge and size differences.

• In vitro kinase assays: Identify kinases that can phosphorylate CAPZA1 and the specific sites involved.

• Mutagenesis studies: Generate phospho-mimetic (e.g., S→D) or phospho-deficient (e.g., S→A) mutants at suspected modification sites to study functional implications.

How can multi-omics approaches be integrated with CAPZA1 antibody-based studies?

To leverage multi-omics with antibody-based CAPZA1 research:

• Proteomics: Combine CAPZA1 immunoprecipitation with mass spectrometry to identify interaction partners under different conditions.

• Transcriptomics: Correlate CAPZA1 protein levels (detected by antibodies) with transcriptomic changes following CAPZA1 knockdown or overexpression.

• ChIP-seq: If CAPZA1 has nuclear functions, perform ChIP-seq using CAPZA1 antibodies to identify genomic binding sites.

• Spatial transcriptomics: Combine CAPZA1 immunostaining with spatial transcriptomics to correlate CAPZA1 protein localization with local gene expression patterns.

• Single-cell analysis: Use CAPZA1 antibodies in single-cell protein analysis methods like CyTOF or CODEX to correlate CAPZA1 expression with cell states.

What are the most promising research directions for understanding CAPZA1's role in cancer development and progression?

Based on current evidence, researchers should consider:

• Prognostic biomarker development: Further investigate CAPZA1 as a potential prognostic marker in gastric cancer, where its overexpression correlates with favorable outcomes .

• Tumor suppressor mechanisms in HCC: Explore the molecular mechanisms by which CAPZA1 inhibits EMT, migration, and invasion in hepatocellular carcinoma .

• Therapeutic targeting: Investigate whether modulating CAPZA1 expression or function could serve as a therapeutic approach in cancers where its loss promotes progression.

• Cancer-specific cytoskeletal dynamics: Study how CAPZA1-regulated actin dynamics differ between normal and cancer cells, potentially revealing new therapeutic vulnerabilities.

• Metastasis regulation: Further investigate how CAPZA1 affects intrahepatic metastasis of HCC cells, as demonstrated in orthotopic transplantation tumor models .