ACTL7B Antibody

What is the biological significance of ACTL7B in reproductive research?

ACTL7B is a member of the actin-related protein (ARP) family that shares significant amino acid sequence identity with conventional actins. It is expressed predominantly in the testis and plays critical roles in spermatogenesis and male fertility . Unlike conventional actins, ACTL7B exhibits specialized functions in reproductive biology, participating in cytoskeletal organization, protein trafficking, and gene regulation during sperm development .

Research using knockout models has demonstrated that ACTL7B deficiency leads to spermatogenic arrest starting from step 9 of spermiogenesis, with abnormal spermatid formation and subsequent degradation . The protein shows both cytoplasmic localization (particularly in round and elongating spermatids) and intranuclear presence, suggesting multifunctional roles in sperm development . This dual localization pattern makes ACTL7B an important target for investigating the molecular mechanisms underpinning male fertility.

How do ACTL7B antibodies differ in their technical specifications?

Several ACTL7B antibodies are available for research applications, each with distinct characteristics that make them suitable for different experimental approaches:

These antibodies target different epitopes of ACTL7B, with some recognizing the N-terminal region (aa 1-100), others targeting internal regions (aa 286-377), and some binding to the full-length protein . This diversity allows researchers to select antibodies that best match their experimental needs, whether for detecting specific domains or studying the protein in different species. The varying applications (Western blot, immunohistochemistry, ELISA) provide flexibility for different experimental approaches.

What is the expression pattern of ACTL7B in normal and pathological tissues?

ACTL7B exhibits a predominantly testis-specific expression pattern in normal tissues. Immunohistochemical studies using ACTL7B antibodies have demonstrated:

• Strong cytoplasmic localization in round spermatids, with expression weakening as spermatids elongate

• Intranuclear localization in spermatocytes, with enriched expression in a pattern consistent with synapsed chromosomal localization

• Dual cytoplasmic and nuclear presence in round spermatids

Western blot analysis using ACTL7B antibodies has detected the protein in human testis tissue, human liver tissue, and mouse testis tissue . Interestingly, immunohistochemistry has also revealed ACTL7B expression in human prostate cancer tissue, suggesting potential expression beyond reproductive tissues in certain pathological conditions .

This distinct expression pattern makes ACTL7B antibodies valuable tools for studying testis-specific processes and potentially for discriminating between different types of male infertility. Comparative proteomics studies have identified ACTL7B among the six proteins with the highest discriminating power between obstructive and non-obstructive azoospermia subtypes .

What are the optimal protocols for ACTL7B antibody use in Western blotting?

For optimal Western blot results with ACTL7B antibodies, the following protocol parameters are recommended based on published research:

Sample preparation:

• Tissue sources: Human testis tissue, human liver tissue, mouse testis tissue (positive controls)

• Protein extraction: Standard SDS-PAGE sample preparation methods

• Loading amount: Typically 20-40 μg total protein per lane

Antibody conditions:

• Primary antibody dilution: 1:500-1:3000 (for Proteintech 13537-1-AP)

• Expected molecular weight: 45 kDa (matches calculated weight based on 415 amino acid sequence)

• Blocking solution: Standard blocking buffer (typically 5% non-fat milk or BSA in TBST)

• Secondary antibody: Species-appropriate HRP-conjugated secondary antibody

Detection and visualization:

• Signal development: Standard ECL detection systems

• Exposure time: Optimize based on signal strength, typically 30 seconds to 5 minutes

Notable considerations:

• In co-immunoprecipitation experiments, ACTL7B may appear as a shifted band around 60 kDa when bound to interacting proteins such as DYNLL1 and DYNLL2

• DYNLL1 and DYNLL2 typically appear as bands of approximately 12 kDa in input samples but shift to around 60 kDa in co-IP eluates when bound to ACTL7B

• Storage of antibody at -20°C maintains stability for one year after shipment

For validation, include both positive controls (testis tissue) and negative controls (non-expressing tissues or ACTL7B knockout samples when available).

How should researchers optimize immunohistochemistry protocols with ACTL7B antibodies?

For successful immunohistochemistry applications using ACTL7B antibodies, researchers should follow these methodological guidelines:

Tissue preparation:

• Fixation: Standard formalin fixation and paraffin embedding

• Sectioning: 4-6 μm thick sections on positively charged slides

• Positive control tissues: Human testis tissue, human prostate cancer tissue

• Negative control: Testis tissue from ACTL7B knockout mice has been validated

Staining protocol:

• Deparaffinization and rehydration: Standard xylene and graded ethanol series

• Antigen retrieval: TE buffer pH 9.0 is recommended; citrate buffer pH 6.0 is an alternative

• Blocking: Serum-based blocking solution appropriate for secondary antibody

• Primary antibody dilution: 1:20-1:200 (for Proteintech 13537-1-AP)

• Incubation conditions: Typically overnight at 4°C or 1-2 hours at room temperature

• Detection system: Compatible with primary antibody host species (e.g., rabbit IgG)

• Counterstaining: Hematoxylin for nuclear visualization

Expected staining pattern:

• Cytoplasmic localization in round and elongating spermatids, with intensity decreasing during spermatid elongation

• Nuclear staining in spermatocytes with a pattern consistent with synapsed chromosomal localization

• In prostate cancer tissue, distinct cytoplasmic staining pattern

Validation criteria:

• Absence of staining in ACTL7B knockout tissue sections confirms specificity

• Consistent staining pattern across different antibodies targeting different epitopes

• Correlation with known expression pattern from mRNA studies

For optimal results, it is recommended to titrate the antibody concentration for each specific tissue type and fixation condition.

What is the recommended approach for co-immunoprecipitation studies involving ACTL7B?

Based on successful published research, the following detailed protocol is recommended for co-immunoprecipitation studies with ACTL7B antibodies:

Reagents and materials:

• Anti-ACTL7B antibody (validated for immunoprecipitation)

• Magnetic beads (e.g., Dynabeads)

• Appropriate lysis buffer (preserving protein-protein interactions)

• Protease inhibitor cocktail

• Wash buffers of varying stringency

• Elution buffer

Procedure:

• Antibody coupling to beads:

  • Couple anti-ACTL7B antibody to magnetic beads following manufacturer's instructions

  • Prepare uncoupled beads as a negative control

• Sample preparation:

  • Harvest tissue (e.g., whole wild-type testes) or cells of interest

  • Homogenize in cold lysis buffer containing protease inhibitors

  • Clear lysate by centrifugation (typically 14,000 × g for 10 minutes at 4°C)

• Immunoprecipitation:

  • Incubate lysate with antibody-coupled beads (typically 2-4 hours at 4°C with rotation)

  • Collect beads using a magnetic stand

  • Wash beads thoroughly with wash buffers of increasing stringency

• Elution and analysis:

  • Elute bound proteins with appropriate elution buffer

  • Analyze eluate by mass spectrometry for discovery approaches

  • Alternatively, perform Western blotting with specific antibodies for targeted detection

Expected results and interpretation:

• ACTL7B appears as a band shifted to around 60 kDa in the eluate (rather than the expected 45 kDa) when bound to interacting proteins

• DYNLL1 and DYNLL2 appear as bands of approximately 60 kDa in the eluate (rather than their expected 12 kDa size)

• These molecular weight shifts confirm protein-protein interactions

Validation approaches:

• Perform reciprocal co-IP using antibodies against identified interaction partners

• Include appropriate negative controls (uncoupled beads, IgG control)

• Confirm specificity by comparing results between wild-type and knockout samples

This approach has successfully identified interactions between ACTL7B and dynein light chains DYNLL1 and DYNLL2, demonstrating its effectiveness for studying ACTL7B protein complexes.

How can ACTL7B antibodies be used to investigate nuclear localization and function?

ACTL7B antibodies have proven invaluable for exploring the recently discovered nuclear functions of this protein. The following experimental approaches can be employed:

Immunofluorescence microscopy for nuclear localization:

• Utilize custom antibodies specific to the N-terminus of ACTL7B for optimal detection of nuclear signal

• Employ super-resolution or confocal microscopy for detailed intranuclear localization

• Co-stain with nuclear markers (DAPI) and synaptonemal complex proteins to confirm chromosomal association

• Compare staining patterns between spermatocytes (showing synapsed chromosomal localization) and round spermatids

Nuclear localization sequence (NLS) investigation:

• Researchers identified two potential NLSs within ACTL7B:

  • One near the end of the N-terminal disordered domain (amino acids K40-K50)

  • Another in a surface-facing α-helix within subdomain 4 (amino acids H250-Y261)

• Experimental studies using YFP-tagged ACTL7B domain constructs revealed the actin-like domain [Leu56-Cys415] was responsible for nuclear localization

• Use ACTL7B antibodies to detect endogenous protein localization and compare with expression constructs

Chromatin association studies:

• Perform chromatin immunoprecipitation (ChIP) using ACTL7B antibodies to identify DNA-binding sites

• Combine with RNA-seq data from ACTL7B knockout models to correlate binding with transcriptional changes

• Investigate associations with chromatin remodeling complexes through co-IP and mass spectrometry

HDAC localization dependency:

• Use immunofluorescence with ACTL7B and HDAC antibodies to study co-localization

• Compare HDAC1 and HDAC3 nuclear localization between wild-type and ACTL7B knockout tissues

• Research has shown loss of intranuclear localization of HDAC1 and HDAC3 in the absence of ACTL7B

These approaches have revealed that ACTL7B plays a role in recruiting or maintaining histone deacetylases in the nucleus, thereby potentially affecting chromatin structure and gene expression during spermatogenesis.

What methodology enables the study of ACTL7B interaction with dynein light chains?

The interaction between ACTL7B and dynein light chains DYNLL1/DYNLL2 represents a critical aspect of its function in spermatogenesis. The following comprehensive methodology has been successfully employed to characterize this interaction:

Co-immunoprecipitation and mass spectrometry:

• Couple anti-ACTL7B antibody to magnetic beads (e.g., Dynabeads)

• Prepare protein extracts from whole wild-type testes

• Perform co-IP and analyze eluate by mass spectrometry

• Filter results by:

  • Excluding contaminating peptides (e.g., keratins)

  • Subtracting peptides identified in "beads only" control

  • Filtering against published bead proteomes to remove non-specific binders

Western blot verification:

• Probe co-IP eluates with antibodies against ACTL7B, DYNLL1, and DYNLL2

• Observe molecular weight shifts:

  • ACTL7B shifts from 45 kDa to around 60 kDa

  • DYNLL1/DYNLL2 shift from 12 kDa to around 60 kDa

• Perform reciprocal co-IP using anti-DYNLL1 and anti-DYNLL2 antibodies

Comparative localization studies:

• Use immunofluorescence with antibodies against ACTL7B, DYNLL1, and DYNLL2

• Compare localization patterns in:

  • Wild-type testis tissue

  • ACTL7B-deficient (Actl7b-/-) testis tissue

• Research has shown that DYNLL1 and DYNLL2 are first detected in step 9 spermatids and become mislocalized when ACTL7B is absent

Functional validation through knockout models:

• Generate or obtain ACTL7B-deficient mice

• Compare sperm development, particularly from step 9 spermatids onward

• Correlate phenotypic abnormalities with changes in DYNLL1/DYNLL2 localization

• Perform proteomic analysis of wild-type vs. knockout tissues to identify additional changes

This multi-faceted approach has established that the interaction between ACTL7B and dynein light chains is essential for proper localization of these proteins during spermiogenesis, and disruption of this interaction likely contributes to the spermatogenic arrest observed in ACTL7B-deficient mice.

How can researchers correlate ACTL7B expression with male fertility phenotypes?

Investigating the relationship between ACTL7B expression and male fertility requires a systematic approach combining molecular, cellular, and physiological methods:

Knockout model characterization:

• Generate or obtain ACTL7B-deficient mice using CRISPR/Cas9-mediated gene editing

• Compare fertility parameters between:

  • Homozygous knockout (Actl7b-/-): Infertile with spermatogenic arrest

  • Heterozygous (Actl7b+/-): Unaffected fertility

  • Wild-type (Actl7b+/+): Normal fertility

• Perform detailed histological analysis of testis sections at different developmental stages

Spermatogenesis phenotype characterization:

• Use ACTL7B antibodies for immunohistochemical analysis of wild-type and heterozygous tissues

• Document the progression of spermatogenesis defects in knockout models:

  • Spermatogenic arrest starting from step 9 of spermiogenesis

  • Abnormal spermatid formation and subsequent phagocytosis/degradation

  • Increased levels of autophagy markers

Molecular and proteomic analysis:

• Perform differential protein abundance analysis:

  • 30 proteins show higher abundance in Actl7b-/- testis

  • Several spermatocyte and spermatid-related proteins show lower abundance

• Proteins higher in abundance include those involved in:

  • Protein or nucleic acid degradation

  • Early apoptosis (ANXA5, CTSB, LGMN, TPP1, ENDOD1)

  • Testis immunoregulation (VCAM-1)

Translational human studies:

• Examine ACTL7B expression in human testicular biopsies from:

  • Normospermic individuals

  • Patients with various forms of male infertility

• Comparative proteomics on human testicular tissue has identified ACTL7B among the six proteins/transcripts with the highest discriminating power between obstructive and non-obstructive azoospermia subtypes

This comprehensive approach has established that mutations in ACTL7B are directly related to male infertility in mice, providing a foundation for translational research in humans and suggesting that ACTL7B antibodies may have diagnostic applications in the clinical evaluation of male infertility.

What validation strategies confirm ACTL7B antibody specificity?

Ensuring antibody specificity is crucial for reliable ACTL7B research. The following comprehensive validation strategies have been effectively employed:

Genetic validation using knockout models:

• Test antibodies on tissues from ACTL7B knockout mice (Actl7b-/-)

• Absence of staining in knockout tissues confirms antibody specificity

• Published research demonstrates no detectable staining in Actl7b-/- testis sections using validated antibodies

Overexpression systems:

• Test antibodies on HEK-293T cells overexpressing ACTL7B with epitope tags (e.g., myc-DDK)

• Include vector-only transfected cells as negative controls

• Western blot should detect bands of expected size (45 kDa) only in ACTL7B-expressing cells

• Immunofluorescence should show appropriate subcellular localization

Recombinant protein controls:

• Use purified recombinant ACTL7B protein as a positive control

• Perform peptide competition assays by pre-incubating antibody with immunizing peptide

• Signal should be blocked or significantly reduced when antibody is pre-absorbed with specific peptide

Multiple antibody concordance:

• Use antibodies targeting different epitopes of ACTL7B:

  • N-terminal domain (aa 1-100)

  • Internal region (aa 286-377)

  • Full-length protein

• Consistent results across different antibodies increase confidence in specificity

• Compare staining patterns and Western blot results between antibodies

Tissue expression pattern verification:

• Confirm strongest expression in testis tissue (known to have highest ACTL7B expression)

• Verify cytoplasmic localization in round and elongating spermatids

• Confirm nuclear localization in spermatocytes with pattern consistent with synapsed chromosomal localization

These validation approaches ensure that observations made using ACTL7B antibodies accurately reflect the biology of this important reproductive protein.

What are the key considerations for optimizing ACTL7B antibody performance?

Optimizing ACTL7B antibody performance requires careful attention to several technical factors:

Western blot optimization:

• Antibody dilution: Begin with manufacturer recommendations (e.g., 1:500-1:3000 for Proteintech 13537-1-AP)

• Protein loading: 20-40 μg total protein; may need adjustment for low-abundance samples

• Transfer conditions: For the 45 kDa ACTL7B protein, standard transfer protocols are effective

• Blocking reagents: 5% non-fat milk or BSA in TBST; optimize if background is problematic

• Incubation time and temperature: Typically 1-2 hours at room temperature or overnight at 4°C

Immunohistochemistry optimization:

• Fixation protocol: Standard formalin fixation; excessive fixation may mask epitopes

• Antigen retrieval methods:

  • Primary recommendation: TE buffer pH 9.0

  • Alternative: Citrate buffer pH 6.0

• Antibody dilution: 1:20-1:200; titrate for optimal signal-to-noise ratio

• Detection systems: Select based on primary antibody host species and desired sensitivity

Immunofluorescence considerations:

• Fixation method: Critical for preserving nuclear architecture

• Permeabilization: Ensure adequate nuclear membrane permeabilization for detecting intranuclear signals

• Blocking of non-specific binding: Use appropriate serum matched to secondary antibody species

• Confocal or super-resolution microscopy: Recommended for detailed intranuclear localization studies

Storage and handling:

• Storage temperature: -20°C (stable for one year after shipment)

• Aliquoting: Not necessary for -20°C storage

• Working solution preparation: Dilute in buffer containing carrier protein (e.g., 1% BSA) to prevent non-specific adsorption

Sample-specific considerations:

• Tissue-specific optimization: May require adjustment of protocols for different tissue types

• Species cross-reactivity: Verify antibody reactivity with your species of interest (human, mouse, rat)

• Fresh vs. archived samples: Older specimens may require modified antigen retrieval methods

Following these optimization guidelines will help researchers obtain reliable and reproducible results when using ACTL7B antibodies in their experimental workflows.

How can researchers troubleshoot common issues with ACTL7B antibody applications?

When working with ACTL7B antibodies, researchers may encounter various technical challenges. The following systematic troubleshooting approaches address common issues:

Western blot troubleshooting:

Immunohistochemistry troubleshooting:

Nuclear localization detection issues:

Co-immunoprecipitation issues:

By systematically addressing these common issues, researchers can optimize their ACTL7B antibody applications and obtain reliable, reproducible results in their studies of this important reproductive protein.

How has recent research expanded our understanding of ACTL7B's nuclear functions?

Recent studies have revealed previously unknown nuclear roles for ACTL7B, dramatically expanding our understanding of this protein's function in reproductive biology:

Discovery of intranuclear localization:
Recent research has confirmed the intranuclear presence of ACTL7B in both spermatocytes and round spermatids . Using custom antibodies specific to the N-terminus of ACTL7B, researchers observed enriched nuclear expression in patterns consistent with synapsed chromosomal localization, suggesting involvement in chromatin-associated processes during meiosis . This finding represents a significant shift from earlier understanding that focused primarily on ACTL7B's cytoplasmic functions.

Nuclear localization sequence characterization:
Analysis of ACTL7B sequences across 42 mammalian species identified a conserved nuclear localization sequence (NLS) within the actin-like domain . Specifically, a surface-facing α-helix within subdomain 4 of ACTL7B (amino acids H250-Y261) appears to mediate nuclear transport. This represents a unique mode of nuclear transport different from conventional actin, which typically requires Cofilin for nuclear entry .

Impact on epigenetic regulators:
One of the most significant discoveries is that ACTL7B influences the nuclear localization of histone deacetylases HDAC1 and HDAC3 . In ACTL7B-deficient models, these histone deacetylases fail to properly localize within the nucleus. As HDACs regulate epigenetic-associated acetylation changes that influence gene expression, this observation provides a mechanistic link between ACTL7B and transcriptional regulation during spermatogenesis.

Chromatin regulatory complexes:
Researchers have proposed that testis-specific ARPs like ACTL7B may participate in chromatin regulation through ARP subunit swapping in canonical chromatin regulatory complexes . This hypothesis expands the functional roles of ARPs in cell biology beyond cytoskeletal regulation and suggests that ACTL7B may contribute to the specialized chromatin remodeling required during spermatogenesis.

Transcriptional impact:
Transcriptomic analysis of ACTL7B-deficient testis has revealed varied transcriptional changes, providing further evidence for ACTL7B's role in gene regulation . The connection to HDAC localization offers a potential mechanism for these observed transcriptional effects, linking ACTL7B's physical presence in the nucleus with functional consequences for gene expression.

These discoveries represent a paradigm shift in our understanding of ACTL7B, establishing it as a multifunctional protein with important roles in both nuclear and cytoplasmic processes during male germ cell development.

What methodological advances have improved ACTL7B protein interaction studies?

Recent methodological innovations have significantly enhanced our ability to study ACTL7B protein interactions:

Advanced co-immunoprecipitation approaches:
Researchers have refined co-immunoprecipitation techniques for ACTL7B by coupling specific antibodies to magnetic beads (Dynabeads) for efficient capture of protein complexes . This approach, combined with careful experimental design including uncoupled beads as controls, has enabled more specific identification of true interaction partners. The method successfully revealed interactions between ACTL7B and dynein light chains DYNLL1 and DYNLL2, demonstrating its effectiveness for detecting physiologically relevant protein complexes .

Improved mass spectrometry filtering strategies:
To reduce false positives in mass spectrometry analysis, researchers have implemented sophisticated filtering approaches:

• Exclusion of common contaminants (e.g., keratins)

• Subtraction of peptides identified in "beads only" controls

• Filtering against published bead proteomes to remove proteins that non-specifically bind to the beads

This multilayered filtering strategy significantly improves the signal-to-noise ratio in interaction studies, increasing confidence in identified protein partners.

Reciprocal co-immunoprecipitation validation:
To confirm the specificity of observed interactions, researchers now routinely perform reciprocal co-immunoprecipitation, pulling down with antibodies against both ACTL7B and its putative partners (e.g., DYNLL1 and DYNLL2) . This bidirectional validation strengthens evidence for true biological interactions and helps eliminate technical artifacts.

Molecular weight shift analysis:
A notable methodological insight from recent studies is the observation that ACTL7B and its interaction partners show characteristic molecular weight shifts in co-IP eluates. Specifically, ACTL7B shifts from its expected 45 kDa to approximately 60 kDa, while DYNLL1/DYNLL2 shift from 12 kDa to around 60 kDa . Recognition of these shifts allows researchers to more confidently identify bound protein complexes in Western blot analysis.

Integration with knockout model validation:
Perhaps the most powerful methodological advance is the integration of protein interaction studies with knockout models. By comparing localization patterns of interaction partners (e.g., DYNLL1/DYNLL2) between wild-type and ACTL7B-deficient tissues, researchers can establish the functional significance of identified interactions . This approach has demonstrated that DYNLL1 and DYNLL2 become mislocalized in the absence of ACTL7B, providing strong evidence for the biological relevance of these interactions.

These methodological advances have not only expanded our knowledge of ACTL7B's protein interaction network but also established a framework for more rigorous protein interaction studies in reproductive biology research.

How does ACTL7B research contribute to understanding male fertility disorders?

Research on ACTL7B has provided significant insights into the molecular mechanisms underlying male fertility disorders:

Direct evidence from knockout models:
Studies using ACTL7B-deficient mice have unequivocally established that mutations in ACTL7B directly cause male infertility . Homozygous knockout males (Actl7b-/-) are completely infertile, while heterozygous males (Actl7b+/-) maintain normal fertility. This finding provides a clear genetic link between ACTL7B function and reproductive capacity, identifying ACTL7B as a critical factor in male fertility.

Cellular mechanisms of infertility:
Detailed characterization of ACTL7B knockout phenotypes has revealed specific cellular mechanisms underlying infertility:

• Spermatogenic arrest starting from step 9 of spermiogenesis

• Abnormal spermatid formation followed by phagocytosis and degradation

• Uncoordinated progression of sperm development processes

• Increased levels of autophagy markers

• Mislocalization of dynein light chains DYNLL1 and DYNLL2

These observations provide a mechanistic framework for understanding how ACTL7B deficiency leads to male infertility.

Diagnostic potential in human infertility:
Comparative proteomics on human testicular tissue has identified ACTL7B among the six proteins/transcripts with the highest discriminating power between obstructive and non-obstructive azoospermia subtypes . This finding suggests that ACTL7B expression analysis could have diagnostic value in differentiating causes of male infertility, potentially guiding treatment decisions.

Newly discovered nuclear roles with fertility implications:
The recent discovery of ACTL7B's nuclear functions, particularly its influence on histone deacetylases HDAC1 and HDAC3, provides a novel connection between epigenetic regulation and male fertility . This finding suggests that some cases of unexplained male infertility might involve disruptions to nuclear processes regulated by ACTL7B, opening new avenues for diagnostic and therapeutic research.

Protein interaction network in spermatogenesis:
Identification of ACTL7B's interaction with dynein light chains DYNLL1 and DYNLL2 has revealed a previously unknown protein network important for spermatogenesis . Disruption of this network through ACTL7B deficiency leads to infertility, highlighting the importance of protein-protein interactions in reproductive function and suggesting that mutations affecting these interactions might contribute to human infertility cases.

These contributions establish ACTL7B research as an important field for understanding male infertility at the molecular level, with potential translational implications for diagnosis and treatment of human reproductive disorders.