ACTR8 Antibody

What is ACTR8 and what functions does it perform in cellular contexts?

ACTR8 (ARP8 Actin-Related Protein 8 Homolog), also known as INO80 complex subunit N, is a nuclear actin-related protein with critical functions in chromatin organization. In humans, the canonical protein consists of 624 amino acid residues with a molecular mass of approximately 70.5 kDa . ACTR8 is primarily localized in the nucleus and chromosomes, where it functions as an integral component of the INO80 chromatin remodeling complex .

The protein plays essential roles in:

• ATP-dependent nucleosome remodeling

• DNA repair mechanisms, particularly in response to double-strand breaks

• Replication processes

• Transcriptional regulation

• Maintaining genomic stability

• Functional organization of mitotic chromosomes

Up to three different isoforms have been reported for this protein, and orthologs have been identified across multiple species including mouse, rat, bovine, frog, zebrafish, chimpanzee, and chicken .

What types of ACTR8 antibodies are available for research applications?

ACTR8 antibodies are available in several configurations tailored to different experimental needs:

When selecting an ACTR8 antibody, researchers should consider which region of the protein is most relevant to their study. For instance, N-terminal antibodies (such as ABIN2791275) recognize the first 50 amino acids, while others target middle regions of the protein .

What are the standard experimental applications for ACTR8 antibodies?

ACTR8 antibodies are employed across several experimental methodologies:

Western Blotting (WB) - The most common application for ACTR8 antibodies, enabling quantification and detection of ACTR8 protein expression in cell and tissue lysates. Typical working dilutions range from 1:500 to 1:2000 .

ELISA - Used for quantitative detection of ACTR8 in solution, often with high sensitivity compared to other protein detection methods .

Immunocytochemistry (ICC) and Immunofluorescence (IF) - Allow visualization of the subcellular localization of ACTR8, particularly important given its nuclear and chromosomal distribution .

Each application requires specific optimization, with Western Blot being the most widely validated application across commercially available ACTR8 antibodies .

What are the optimal storage and handling conditions for ACTR8 antibodies?

To maintain antibody function and specificity, the following storage and handling recommendations should be observed:

• Store at -20°C in small aliquots to avoid repeated freeze-thaw cycles

• Most ACTR8 antibodies are supplied in buffer containing PBS (pH 7.4) with preservatives such as sodium azide (0.09%) and stabilizers like glycerol (often 50%)

• Some formulations contain additional preservatives such as Proclin-300 (0.03%)

• For short-term use (up to 1 month), 4°C storage may be acceptable

• Allow antibodies to equilibrate to room temperature before opening vials

• Use sterile technique when handling to prevent contamination

Researchers should note that sodium azide, commonly used as a preservative, is toxic and hazardous. It also inhibits horseradish peroxidase activity and should not be used in HRP-conjugated detection systems .

How specific are ACTR8 antibodies, and what is their cross-reactivity profile?

ACTR8 antibodies vary in their specificity and cross-reactivity profiles:

Most commercially available antibodies demonstrate high specific reactivity to human ACTR8. Cross-reactivity with orthologs from other species depends on sequence conservation. For instance, some antibodies like ABIN2791275 show predicted reactivity to:

• Cow: 100%

• Dog: 100%

• Guinea Pig: 100%

• Human: 100%

• Mouse: 100%

• Rabbit: 100%

• Rat: 100%

How can Western blot protocols be optimized for reliable ACTR8 detection?

Optimizing Western blot for ACTR8 detection requires several specific considerations:

Sample Preparation:

• Nuclear extraction protocols are crucial as ACTR8 is predominantly nuclear

• Use phosphatase and protease inhibitors to prevent degradation

• Sonication may improve extraction efficiency of chromatin-bound ACTR8

Gel Selection and Transfer:

• Use 8-10% acrylamide gels appropriate for the 70.5 kDa size of ACTR8

• Semi-dry transfer at lower voltage (15V) for longer duration (60 minutes) often yields better results for nuclear proteins

Blocking and Antibody Incubation:

• 5% BSA in TBST is generally preferable to milk-based blocking for nuclear proteins

• Primary antibody dilution typically ranges from 1:500 to 1:2000

• Overnight incubation at 4°C often improves signal quality

• Extensive washing (5+ washes of 5 minutes each) minimizes background

Controls and Validation:

• Include positive control lysates from cells known to express ACTR8

• Consider using recombinant ACTR8 as a standard

• Include loading controls specific for nuclear proteins (e.g., Lamin B1)

When troubleshooting, verify the expected molecular weight (70.5 kDa for the canonical form) and be aware that post-translational modifications may affect migration patterns. For low-abundance detection, signal enhancement systems or highly sensitive chemiluminescence reagents may be necessary .

What are effective validation strategies for confirming ACTR8 antibody specificity?

Rigorous validation is essential for ensuring the reliability of ACTR8 antibody-based experiments:

Primary Validation Methods:

• Genetic Validation: Compare wild-type cells with ACTR8 knockout/knockdown samples to confirm antibody specificity

• Peptide Competition Assay: Pre-incubate antibody with immunizing peptide to validate signal specificity

• Orthogonal Detection: Confirm results using multiple antibodies targeting different epitopes of ACTR8

• Mass Spectrometry Validation: Confirm identity of immunoprecipitated protein bands

Secondary Validation Approaches:

• Immunoprecipitation followed by Western blot (IP-WB)

• Expression correlation with mRNA levels across tissues

• Subcellular localization confirmation through fractionation

• Co-localization with known interaction partners (e.g., other INO80 complex components)

The validation method should match the intended application. For instance, an antibody that performs well in Western blot may not necessarily work in immunofluorescence applications. Document all validation steps methodically, including batch/lot information, as antibody performance can vary between lots .

How can researchers troubleshoot inconsistent results when using ACTR8 antibodies?

When encountering variability in ACTR8 antibody performance, consider this systematic troubleshooting approach:

For Western Blotting Issues:

• Multiple Bands: May indicate isoforms (up to 3 reported for ACTR8), degradation products, or post-translational modifications

• Weak Signal: Try increasing antibody concentration, extending incubation time, or using more sensitive detection reagents

• High Background: Increase blocking time, use more stringent washing, or try different blocking reagents

For Immunofluorescence/ICC Issues:

• Non-specific Staining: Adjust fixation method (try both paraformaldehyde and methanol fixation)

• Weak Nuclear Signal: Try antigen retrieval methods appropriate for nuclear proteins

• Inconsistent Cell-to-Cell Staining: Consider cell cycle-dependent expression of ACTR8

General Troubleshooting Steps:

• Verify antibody storage conditions and avoid freeze-thaw cycles

• Test multiple antibody lots if possible

• Include appropriate positive and negative controls

• Optimize protein extraction for nuclear proteins

• Consider cell type-specific expression levels and regulation

Document all experimental variables systematically when troubleshooting, including cell types, fixation methods, buffer compositions, and incubation conditions to identify sources of variability .

What considerations are important when designing chromatin immunoprecipitation (ChIP) experiments with ACTR8 antibodies?

ACTR8's role in chromatin remodeling makes ChIP a valuable technique for studying its genomic interactions, but requires specific optimization:

Cross-linking and Chromatin Preparation:

• Dual cross-linking (using both formaldehyde and protein-specific cross-linkers) may improve yield for chromatin-remodeling proteins

• Sonication parameters should be carefully optimized to generate 200-500bp fragments

• Include protocols to enrich for nuclear fractions prior to sonication

IP Conditions:

• Pre-clearing with protein A/G beads is essential to reduce background

• Higher antibody concentrations may be needed compared to typical transcription factors

• Extended incubation times (overnight to 24 hours) often improve chromatin capture

Controls and Analysis:

• Input controls are critical for normalization

• IgG negative controls should match the host species of the ACTR8 antibody

• Positive controls should target regions where ACTR8/INO80 complex is known to bind

• Sequential ChIP (re-ChIP) can confirm co-localization with other INO80 complex components

Data Interpretation:

• ACTR8, as part of the INO80 complex, may show broad rather than sharp peaks

• Analysis should focus on enrichment at promoters, enhancers, and sites of DNA damage

• Integration with histone modification data can provide functional context

When analyzing ChIP-seq data, consider that ACTR8's genomic distribution may change following cellular stresses, particularly DNA damage, reflecting its role in DNA repair mechanisms .

How can ACTR8 antibodies be effectively used to study the INO80 chromatin remodeling complex?

Investigation of ACTR8 within the INO80 complex requires specialized approaches:

Co-Immunoprecipitation (Co-IP) Studies:

• Use ACTR8 antibodies as bait to pull down the entire INO80 complex

• Validate interactions with other complex components (e.g., INO80, ACTR5, YY1)

• Consider native versus cross-linked protocols depending on interaction strength

• Use appropriate detergents that maintain nuclear protein interactions

Functional Analyses:

• Combine immunodepletion with in vitro nucleosome remodeling assays

• Use ACTR8 antibodies in chromatin accessibility assays (e.g., ATAC-seq with antibody-mediated depletion)

• Employ proximity ligation assays to confirm in situ interactions with other complex members

Localization Studies:

• Use super-resolution microscopy to map ACTR8 to specific chromatin domains

• Perform ChIP-seq targeting ACTR8 alongside other INO80 components to identify complex binding sites

• Analyze co-occupancy with histone variants (particularly H2A.Z) that are exchanged by INO80

Methodological Table: Approaches for Studying ACTR8 in the INO80 Complex

When interpreting results, remember that ACTR8's functions may extend beyond the INO80 complex, so not all ACTR8-dependent effects necessarily reflect INO80 activity .

What methodologies are optimal for studying ACTR8's role in DNA repair mechanisms?

ACTR8's involvement in DNA repair processes can be investigated using several complementary approaches:

DNA Damage Response Assays:

• Immunofluorescence to detect ACTR8 recruitment to DNA damage sites (look for co-localization with γH2AX)

• Live-cell imaging with fluorescently tagged ACTR8 to track recruitment kinetics

• ChIP-qPCR at engineered DNA break sites to quantify ACTR8 enrichment

Functional Assessment:

• Measure DNA repair efficiency in cells with ACTR8 depletion/inhibition

• Use comet assays to assess DNA damage resolution

• Monitor homologous recombination and non-homologous end joining repair pathways

Interaction Studies:

• Identify damage-specific interactions using ACTR8 antibodies for immunoprecipitation after DNA damage induction

• Compare ACTR8 complex composition in damaged versus undamaged conditions

• Assess post-translational modifications of ACTR8 following DNA damage

Experimental Design Considerations:

• Include appropriate DNA damage-inducing agents (e.g., ionizing radiation, etoposide, neocarzinostatin)

• Use time-course experiments to capture early and late recruitment dynamics

• Compare different damage types (double-strand breaks, single-strand breaks, UV damage)

ACTR8 antibodies with high specificity and low background are critical for these applications, as DNA damage response proteins often form discrete nuclear foci that can be difficult to distinguish from non-specific staining .

What are the implications of ACTR8 dysregulation in genomic disorders and cancer research?

ACTR8 dysregulation has significant implications for genomic stability and disease development:

Cancer Research Applications:

• Use ACTR8 antibodies to assess expression levels across tumor types

• Correlate ACTR8 expression with genomic instability markers

• Investigate ACTR8's role in chromatin accessibility changes in cancer cells

Mechanisms of Dysregulation:

• Altered expression levels affecting INO80 complex function

• Mutations affecting protein-protein interactions

• Changes in subcellular localization

Potential Research Directions:

• Prognostic value of ACTR8 expression in tumor samples

• Correlation with treatment response, particularly to DNA-damaging therapies

• Synthetic lethality approaches targeting cells with ACTR8 dysfunction

Methodological Approaches:

• Tissue microarray analysis with ACTR8 antibodies

• Correlation of ACTR8 expression with clinical outcomes

• Integration with genomic instability signatures

Researchers investigating ACTR8 in disease contexts should consider both loss-of-function and gain-of-function scenarios, as either may contribute to pathological states through disruption of normal chromatin dynamics and DNA repair functions .

How can ACTR8 antibodies be effectively used in fluorescence microscopy to study nuclear localization?

Fluorescence microscopy offers valuable insights into ACTR8's nuclear distribution and dynamics:

Sample Preparation:

• Test multiple fixation methods (4% paraformaldehyde, methanol, or combinations)

• Permeabilization is critical - use 0.1-0.5% Triton X-100 for nuclear proteins

• Consider epitope accessibility - some fixation methods may mask nuclear epitopes

Antibody Selection and Validation:

• Choose antibodies specifically validated for immunofluorescence applications

• Compare multiple antibodies targeting different epitopes

• Verify specificity using knockdown/knockout controls

Advanced Imaging Techniques:

• Super-resolution microscopy (STED, STORM, SIM) can reveal subnuclear distribution

• Live-cell imaging with tagged constructs can complement antibody-based approaches

• FRAP (Fluorescence Recovery After Photobleaching) can assess ACTR8 dynamics

Co-localization Studies:

• Use markers for nuclear compartments (nucleolus, nuclear speckles, etc.)

• Co-stain with other INO80 complex components

• Examine co-localization with DNA damage markers before and after damage induction

Quantitative Analysis:

• Measure nuclear/cytoplasmic distribution ratios

• Quantify focal accumulation in response to DNA damage

• Analyze correlation with chromatin density markers (DAPI intensity)

For optimal results, combine antibody-based detection with orthogonal approaches like fluorescently-tagged ACTR8 expression to confirm localization patterns and study dynamic behavior .

What considerations are important when using ACTR8 antibodies in different model organisms?

Working with ACTR8 across different model organisms presents both challenges and opportunities:

Cross-Reactivity Assessment:

• Sequence homology varies between species, affecting antibody recognition

• Some antibodies show broad cross-reactivity (human, mouse, rat, cow, dog)

• Species-specific antibodies exist for some models (e.g., zebrafish)

Validation in Non-Human Models:

• Western blot validation is essential before proceeding to other applications

• Species-appropriate positive controls should be included

• Consider tissue-specific expression patterns that may differ between species

Model-Specific Considerations:

Experimental Adaptations:

• Adjust tissue fixation protocols based on organism-specific tissue architecture

• Modify extraction buffers for different cellular compositions

• Consider developmental timing of ACTR8 expression, which may vary between species

When working with less common model organisms, consider developing custom antibodies against species-specific ACTR8 sequences if commercial options show poor cross-reactivity .

Future Directions in ACTR8 Antibody Research

As research on chromatin remodeling and nuclear organization advances, ACTR8 antibodies will continue to be valuable tools. Emerging directions include:

• Development of conformation-specific antibodies that distinguish ACTR8 in different complex states

• Antibodies recognizing post-translational modifications specific to DNA damage response

• Humanized antibodies for potential therapeutic applications targeting chromatin remodeling in disease

• Integration with new technologies like spatial transcriptomics to link ACTR8 localization with gene expression

Recommendations for Optimal ACTR8 Antibody Usage

For researchers working with ACTR8 antibodies, the following best practices are recommended: