ACTR1B Antibody

What is ACTR1B and what cellular functions does it perform?

ACTR1B (also known as Beta-centractin, Actin-related protein 1B, or ARP1B) is a component of a multi-subunit complex involved in microtubule-based vesicle motility. It is primarily associated with the centrosome . The ACTR1B protein ensures proper cellular responses to signaling cues that can impact cell fate and behavior through various pathways. Its effective functioning supports cellular responses to external stimuli and environmental changes . ACTR1B is structurally related to actin and plays an important role in intracellular transport mechanisms, particularly those associated with the dynactin complex.

What are the common applications for ACTR1B antibodies in research?

ACTR1B antibodies are validated for multiple research applications including Western Blotting (WB), Immunohistochemistry (IHC), Immunocytochemistry/Immunofluorescence (ICC/IF), Immunoprecipitation (IP), and Flow Cytometry (Flow Cyt) . These applications allow researchers to detect, localize, and quantify ACTR1B in various experimental contexts. Western blotting is particularly useful for identifying ACTR1B protein expression levels in different cell types, while immunohistochemistry helps visualize its distribution in tissue sections. Immunoprecipitation can be employed to study protein-protein interactions involving ACTR1B, providing insights into its functional networks within the cell.

Which cell lines are commonly used for validating ACTR1B antibodies?

Several human cell lines have been validated for ACTR1B antibody reactivity, including:

• Jurkat (Human T cell leukemia cells from peripheral blood)

• MOLT-4 (Human lymphoblastic leukemia cell line)

• HeLa (Human epithelial cells from cervix adenocarcinoma)

• HepG2 (Human liver hepatocellular carcinoma)

• 293 (Human epithelial cells from embryonic kidney)

These cell lines express detectable levels of ACTR1B and can serve as positive controls in various experimental setups. When establishing a new experimental system, researchers should consider using one of these validated cell lines as a reference point.

How can cross-reactivity between ACTR1A and ACTR1B be managed in experimental designs?

Some antibodies, such as EPR16968(B) (ab203833), recognize both ACTR1B and ACTR1A proteins . When experimental questions require specific detection of ACTR1B without ACTR1A interference, researchers should:

• Select antibodies specifically validated for exclusive ACTR1B detection, such as EPR16969 (ab203835/ab251406)

• Implement additional controls using ACTR1A-specific knockdown or knockout systems

• Validate results with secondary methods like mass spectrometry

• Consider using epitope-specific antibodies targeting regions with lower sequence homology

What blocking and dilution conditions optimize ACTR1B antibody performance in Western blotting?

Optimal blocking and dilution conditions for ACTR1B antibodies in Western blotting typically involve:

• Blocking buffer: 5% Non-Fat Dry Milk (NFDM) in TBST

• Primary antibody dilution: 1/1000 to 1/10000, depending on the specific antibody

• Secondary antibody: Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/1000 dilution

These conditions minimize background while maximizing specific signal detection. For particularly challenging samples or when background issues persist, researchers might consider alternative blocking agents such as BSA or optimization of antibody incubation times and temperatures. Extended washing steps with TBST can also improve signal-to-noise ratios in Western blot applications.

How can researchers validate ACTR1B antibody specificity in new experimental systems?

To validate ACTR1B antibody specificity in a new experimental system, researchers should implement a multi-pronged approach:

• Positive controls: Include validated cell lines known to express ACTR1B (e.g., MOLT-4, HeLa)

• Negative controls: Utilize ACTR1B knockdown/knockout samples generated through siRNA or CRISPR-Cas9

• Peptide competition assay: Pre-incubate the antibody with immunizing peptide to confirm specific binding

• Cross-species validation: If using the antibody in a new species, confirm reactivity based on sequence homology and empirical testing

• Multiple detection methods: Validate findings using different antibody clones or alternative detection methods

This comprehensive validation approach ensures reliable and reproducible results when introducing ACTR1B antibodies into new experimental contexts or model systems.

What are the critical considerations when using ACTR1B antibodies for immunohistochemistry?

When employing ACTR1B antibodies for immunohistochemistry (IHC), researchers should consider:

• Fixation protocol: Different antibody clones may perform optimally with specific fixation methods (formaldehyde, paraformaldehyde, alcohol-based)

• Antigen retrieval: Heat-induced epitope retrieval (HIER) or enzymatic methods may be necessary to unmask epitopes

• Endogenous peroxidase quenching: For HRP-based detection systems

• Background reduction: Tissue-specific blocking strategies to minimize non-specific binding

• Sensitivity threshold: Enhanced detection systems for low-abundance targets

• Controls: Include positive control tissues and negative controls (primary antibody omission, isotype controls)

The EPR16969 antibody clone has been validated for IHC-P (paraffin-embedded) applications with human samples , providing a reliable starting point for ACTR1B immunohistochemical studies.

What strategies can address weak or absent ACTR1B signal in Western blotting?

When encountering weak or absent ACTR1B signals in Western blotting, researchers can implement the following strategies:

• Sample preparation optimization:

  • Ensure complete lysis with appropriate detergents

  • Include protease inhibitors to prevent degradation

  • Optimize protein loading (10-20 μg total protein recommended)

• Technical adjustments:

  • Increase antibody concentration (e.g., from 1/10000 to 1/1000)

  • Extend primary antibody incubation time (overnight at 4°C)

  • Enhance detection sensitivity with amplification systems

• Blocking and washing optimization:

  • Test alternative blocking buffers (BSA vs. NFDM)

  • Adjust washing stringency and duration

• Expression verification:

  • Confirm ACTR1B expression in your samples via RT-PCR or other methods

  • Consider using positive control lysates from validated cell lines (Jurkat, MOLT-4, HeLa)

These systematic approaches address the most common causes of detection failure and enable successful ACTR1B visualization in Western blot applications.

How can researchers distinguish between specific and non-specific bands when using ACTR1B antibodies?

Distinguishing between specific and non-specific bands requires a methodical approach:

• Molecular weight verification: Compare observed bands to the predicted ACTR1B molecular weight (approximately 42 kDa)

• Positive controls: Include lysates from cell lines with confirmed ACTR1B expression (e.g., Jurkat, MOLT-4, HeLa)

• Validation experiments:

  • siRNA knockdown: Specific bands should decrease in intensity

  • Overexpression systems: Specific bands should increase in intensity

  • Peptide competition: Pre-incubation with immunizing peptide should eliminate specific bands

• Multiple antibody validation: Test different antibody clones targeting distinct epitopes

• Loading controls: Ensure equal loading across samples to properly compare band intensities

This comprehensive approach provides multiple lines of evidence for band specificity, enhancing confidence in experimental interpretations.

What factors influence ACTR1B antibody cross-reactivity across species?

Cross-species reactivity of ACTR1B antibodies depends on several factors:

• Epitope conservation: Higher sequence homology in the antibody-binding region increases cross-reactivity potential

• Documented cross-reactivity: Several ACTR1B antibodies have demonstrated reactivity across species:

  • Human, Mouse, Rat (most common)

  • Extended reactivity to Dog, Guinea Pig, Rabbit, Monkey, Bat

  • Some antibodies show broader reactivity including Cow, Horse, Pig, and Zebrafish

• Epitope targeting: Antibodies targeting highly conserved functional domains typically show broader cross-reactivity

• Validation requirement: Cross-reactivity predictions based on sequence homology should be empirically validated

When planning cross-species experiments, researchers should prioritize antibodies with documented reactivity in their species of interest or validate antibodies in their specific experimental system.

How can ACTR1B antibodies be employed in studying centrosome dynamics and vesicular transport?

ACTR1B antibodies can be powerful tools for investigating centrosome dynamics and vesicular transport through:

• Co-localization studies: Combining ACTR1B antibodies with markers for centrosomes, microtubules, or vesicular compartments in immunofluorescence microscopy reveals spatial relationships and potential functional interactions

• Live-cell imaging: Using fluorescently tagged anti-ACTR1B antibody fragments to track dynactin complex movements in real-time

• Super-resolution microscopy: Employing techniques like STORM or PALM with ACTR1B antibodies to visualize nanoscale organization within the centrosome

• Immunoprecipitation (IP): Identifying ACTR1B-interacting proteins involved in centrosome function and vesicular transport

• Proximity ligation assays: Detecting in situ interactions between ACTR1B and other components of the vesicular transport machinery

These techniques provide complementary approaches to understanding ACTR1B's role in coordinating centrosome-associated functions and microtubule-based vesicle motility.

What are the considerations for developing multiplex assays incorporating ACTR1B antibodies?

When developing multiplex assays incorporating ACTR1B antibodies, researchers should address:

• Antibody compatibility:

  • Ensure primary antibodies originate from different host species to avoid cross-reactivity

  • Verify that secondary antibodies specifically recognize their intended primary antibody

• Spectral separation:

  • Select fluorophores with minimal spectral overlap

  • Implement appropriate controls to correct for bleed-through

• Fixation optimization:

  • Identify fixation protocols compatible with all target epitopes

  • Consider sequential immunostaining for sensitive epitopes

• Validation with single-plex controls:

  • Compare multiplex results with single-antibody staining to confirm signal specificity

  • Include appropriate blocking steps between sequential antibody applications

• Quantification strategies:

  • Develop consistent image acquisition parameters

  • Implement appropriate analysis algorithms for co-localization or expression correlation

These considerations ensure reliable and interpretable results when incorporating ACTR1B antibodies into complex multiplex experimental designs.

How can researchers employ ACTR1B antibodies in studying disease mechanisms?

ACTR1B antibodies can be valuable tools for investigating disease mechanisms through:

• Expression profiling: Comparing ACTR1B levels across normal and pathological samples to identify alterations associated with disease states

• Subcellular localization analysis: Detecting abnormal ACTR1B distribution that might indicate disrupted vesicular transport or centrosome dysfunction

• Post-translational modification assessment: Using modification-specific antibodies to examine how disease conditions affect ACTR1B regulation

• Protein interaction studies: Employing co-immunoprecipitation with ACTR1B antibodies to identify altered protein interactions in disease contexts

• Therapeutic target validation: Evaluating the consequences of ACTR1B modulation in disease models to assess its potential as a therapeutic target

These approaches can provide insights into how alterations in ACTR1B function contribute to disease pathogenesis, particularly in conditions involving disrupted intracellular transport or centrosome abnormalities.

How can ACTR1B antibodies be integrated into proximity labeling approaches?

Proximity labeling techniques combining ACTR1B antibodies with enzymatic tags offer powerful approaches for studying protein interactions and microenvironments:

• Antibody-enzyme fusion constructs: Engineering ACTR1B antibodies fused to enzymes like BioID, APEX2, or TurboID for proximity-dependent labeling

• Methodological workflow:

  • Introduce the antibody-enzyme fusion to live or fixed cells

  • Activate the enzyme to label proteins in close proximity to ACTR1B

  • Purify and identify labeled proteins via mass spectrometry

• Applications:

  • Mapping the ACTR1B interactome at specific subcellular locations

  • Identifying transient or weak interactions missed by traditional co-immunoprecipitation

  • Characterizing the dynamics of ACTR1B associations during cellular processes

• Technical considerations:

  • Validate that the antibody-enzyme fusion retains specific binding to ACTR1B

  • Optimize labeling conditions to balance specificity and coverage

  • Include appropriate controls to distinguish specific interactions from background

This emerging approach extends beyond conventional antibody applications to provide spatial and temporal information about ACTR1B's molecular neighborhood.

What are the considerations for using ACTR1B antibodies in single-cell protein analysis techniques?

Single-cell protein analysis with ACTR1B antibodies presents unique challenges and opportunities:

• Mass cytometry (CyTOF):

  • Conjugate ACTR1B antibodies with rare earth metals

  • Optimize staining protocols for detection sensitivity

  • Develop appropriate gating strategies for ACTR1B-positive populations

• Single-cell Western blotting:

  • Establish lysis conditions that preserve ACTR1B integrity

  • Optimize antibody concentration and incubation parameters for microfluidic platforms

  • Validate detection specificity with control cell populations

• Imaging mass cytometry/MIBI:

  • Select metal tags compatible with multiplexed imaging

  • Develop tissue preparation protocols that maintain ACTR1B epitope accessibility

  • Implement computational tools for spatial analysis of ACTR1B expression

• Methodological validation:

  • Compare single-cell results with bulk measurements

  • Establish appropriate thresholds for positive signal detection

  • Account for technical variation in single-cell platforms

These emerging techniques enable researchers to examine ACTR1B expression heterogeneity across cell populations and investigate its relationship to cellular phenotypes at unprecedented resolution.

How might engineered antibody formats enhance ACTR1B research capabilities?

Engineered antibody formats offer expanded capabilities for ACTR1B research:

• Nanobodies and single-domain antibodies:

  • Smaller size enables access to sterically hindered epitopes

  • Superior tissue penetration for in vivo imaging

  • Reduced immunogenicity for therapeutic applications

• Bispecific antibodies:

  • Simultaneous targeting of ACTR1B and interacting partners

  • Enhanced specificity for particular ACTR1B complexes

  • Creation of artificial proximity between ACTR1B and effector molecules

• Antibody fragments (Fab, scFv):

  • Reduced steric hindrance for super-resolution microscopy

  • Improved penetration in densely packed cellular structures

  • More controlled epitope targeting

• Implementation considerations:

  • Validate specificity and affinity of engineered formats

  • Optimize conjugation chemistry for labeling applications

  • Develop appropriate expression and purification protocols

These engineered formats extend the traditional capabilities of ACTR1B antibodies, enabling novel experimental approaches and potentially overcoming limitations of conventional antibodies.