ZSWIM6 Antibody

What is ZSWIM6 and what cellular functions does it perform?

ZSWIM6 (also known as KIAA1577) is a zinc finger SWIM domain-containing protein that functions as a chromatin regulator involved in nervous system development . It plays a crucial role in striatal morphology and motor regulation . The protein contains zinc finger motifs that facilitate interactions with DNA and other proteins . ZSWIM6 predominantly localizes to the nucleus where it associates with repressive chromatin regulators, linking gene regulation with synapse and circuit function during striatal development . Knockout studies in mice have demonstrated that ZSWIM6 deficiency results in reduced cortical and striatal volumes and alterations in the number and structure of medium spiny neurons in the striatum .

Which antibodies are available for ZSWIM6 detection and what applications are they validated for?

Several antibodies are available for ZSWIM6 detection, with ab122301 being one well-documented example. This rabbit polyclonal antibody targets an immunogen corresponding to a recombinant fragment within human ZSWIM6 amino acids 600-700 . It has been validated for:

• Immunohistochemistry on paraffin-embedded tissues (IHC-P): Successful detection in human smooth muscle tissue at 1/75 dilution

• Immunocytochemistry/immunofluorescence (ICC/IF): Shows positivity in the cytoplasm of human A-431 cells at recommended concentrations of 1-4 μg/ml

The antibody is reactive with human samples and has been cited in published research .

How should sample preparation be optimized for ZSWIM6 immunodetection?

For optimal ZSWIM6 immunodetection, sample preparation depends on the application:

For immunofluorescence:

• Fix cells with paraformaldehyde (PFA) and permeabilize with Triton X-100

• When using cell lines such as A-431, cytoplasmic staining can be observed

For immunohistochemistry:

• Use paraffin embedding for tissue samples (demonstrated successful with human smooth muscle tissue)

• Apply the antibody at appropriate dilution (1/75 has been validated)

When considering subcellular localization studies, it's important to note that approximately 75% of ZSWIM6 protein is found in the nucleus of cultured neurons . This nuclear localization is dependent on a nuclear localization signal (NLS), as deletion of this sequence abolishes nuclear enrichment .

What precautions should be taken to ensure ZSWIM6 antibody specificity?

To ensure ZSWIM6 antibody specificity:

• Include appropriate positive controls: Use tissues or cell lines with known ZSWIM6 expression such as neuronal tissues or A-431 cells

• Include negative controls:

  • Primary antibody omission

  • Use of tissues from ZSWIM6 knockout models as negative controls

  • Competitive blocking with the immunizing peptide

• Validate subcellular localization: Confirm that staining patterns align with expected localization (approximately 75% nuclear in neurons)

• Cross-validate with other methods: Combine antibody-based detection with techniques like RT-PCR or Western blotting

• Consider activity-dependent changes: Be aware that certain conditions like picrotoxin (PTX) exposure can decrease nuclear ZSWIM6 localization

How can ZSWIM6 antibodies be used to investigate chromatin regulation mechanisms?

ZSWIM6 functions as a chromatin regulator, and its antibodies can be instrumental in investigating these mechanisms through several approaches:

• Chromatin Immunoprecipitation (ChIP): ZSWIM6 antibodies can be used to identify genomic sites where ZSWIM6 binds. Research indicates that ZSWIM6 associates with repressive chromatin regulators, making ChIP a valuable technique for mapping its genomic targets .

• Co-Immunoprecipitation (Co-IP): ZSWIM6 has been shown to interact with several chromatin-associated proteins including SUZ12, HDAC1, and histone H3 . Researchers can use ZSWIM6 antibodies for Co-IP experiments to:

  • Validate known interactions

  • Discover novel protein partners

  • Investigate how these interactions change under different physiological conditions

• Chromatin Accessibility Studies: Conditional knockout of ZSWIM6 results in broad increases in chromatin accessibility as determined by ATAC-seq, consistent with its role as a repressive chromatin regulator . ZSWIM6 antibodies can be used in conjunction with techniques like ChIP-seq to correlate ZSWIM6 binding with changes in chromatin state.

• Activity-Dependent Regulation: Since neuronal activity (induced by picrotoxin) appears to alter ZSWIM6 nuclear localization, antibodies can be used to track these changes through immunofluorescence studies under various activity conditions .

What methodological approaches can be used to study ZSWIM6 in neurodevelopmental disorders?

Given ZSWIM6's association with neurodevelopmental disorders, several methodological approaches utilizing ZSWIM6 antibodies can be employed:

• Comparative Expression Analysis:

  • Compare ZSWIM6 expression levels and patterns in postmortem brain samples from individuals with intellectual disability, autism, or schizophrenia versus controls

  • The recurrent de novo nonsense variant (c.2737C>T [p.Arg913Ter]) in ZSWIM6 causes severe intellectual disability and neurological features

• Variant Protein Detection:

  • Design antibodies that can distinguish between wild-type ZSWIM6 and truncated variants like p.Arg913Ter

  • Research shows that the c.2737C>T variant does not trigger nonsense-mediated decay, suggesting a truncated protein is produced

• Functional Studies in Model Systems:

  • Use antibodies to validate CRISPR-engineered cellular models expressing ZSWIM6 variants

  • Immunohistochemistry to characterize Zswim6 knockout mice, which display:

    • Reduced cortical and striatal volumes

    • Altered dendritic complexity and spine density of striatal neurons

    • Multiple repetitive motor abnormalities

• Circuit-Level Investigations:

  • Combine ZSWIM6 immunostaining with markers of specific neural circuits

  • Focus on striatal development, as ZSWIM6 is important for striatal morphology and motor regulation

How can ZSWIM6 antibodies be used to investigate its interactions with the ubiquitin pathway?

ZSWIM6 has a postulated role in the ubiquitin pathway, supported by pathological findings in patients with ZSWIM6 variants that resemble disorders of ubiquitination . To investigate this connection:

• Co-localization Studies:

  • Use ZSWIM6 antibodies in combination with antibodies against ubiquitin pathway components

  • Examine if ZSWIM6 co-localizes with ubiquitin or ubiquitin-related proteins in neuronal cells

• Biochemical Analysis:

  • Employ ZSWIM6 antibodies for immunoprecipitation followed by ubiquitin western blotting to detect ubiquitinated ZSWIM6

  • Investigate if ZSWIM6 itself is subject to ubiquitination-dependent regulation

• Neuropathological Examination:

  • Compare ZSWIM6-deficient neurons with those from known ubiquitination disorders

  • Nerve conduction studies and sural nerve biopsies of patients with ZSWIM6 variants show similarities to disorders of ubiquitination such as giant axonal neuropathy 1 (GAN1)

  • Light and electron microscopy reveal reduced myelinated fiber density and abnormal accumulation of neurofilaments, similar to ubiquitination disorders

• Substrate Identification:

  • Use proximity labeling combined with ZSWIM6 antibodies to identify potential ubiquitination substrates that interact with ZSWIM6

What experimental designs are recommended for studying ZSWIM6 across developmental stages?

ZSWIM6 exhibits developmentally regulated expression patterns in the brain, making temporal studies crucial. The following experimental designs are recommended:

• Developmental Expression Profiling:

  • Use ZSWIM6 antibodies for immunohistochemistry on brain sections at different developmental stages

  • In zebrafish larvae, zswim6 is expressed in regions of the telencephalon, midbrain, hindbrain, and retina

  • In embryonic mice, Zswim6 is initially expressed in ganglionic eminences and subsequently in the cortical plate, developing amygdala, and portions of the thalamus and hypothalamus

  • Postnatally, telencephalic expression becomes more restricted to the striatum

• Conditional Knockout Analysis:

  • Use Cre-lox systems for stage-specific deletion, such as the Dlx5/6-Cre line that expresses Cre in ventral GABAergic progenitors from embryonic day 12.5

  • Validate recombination via in situ hybridization across the floxed exon

  • Use ZSWIM6 antibodies to confirm protein loss in targeted regions

• Chromatin Dynamic Studies:

  • Perform ATAC-seq or ChIP-seq at different developmental timepoints

  • Compare chromatin accessibility patterns between wild-type and ZSWIM6-deficient tissues

  • Conditional knockout mice show broad increases in chromatin accessibility

• Behavioral Correlations:

  • Link developmental ZSWIM6 expression patterns to the emergence of motor and cognitive phenotypes

  • Zswim6 knockout mice display motor abnormalities and increased sensitivity to amphetamine

What are common pitfalls when using ZSWIM6 antibodies and how can they be addressed?

When working with ZSWIM6 antibodies, researchers may encounter several challenges:

• Dual Localization Patterns:

  • ZSWIM6 shows both nuclear (predominant, ~75%) and cytoplasmic localization in neurons

  • Subcellular fractionation should be performed to confirm immunostaining results

  • When investigating localization, consider that activity modulation with picrotoxin can decrease nuclear ZSWIM6

• Variant-Specific Detection:

  • The p.Arg913Ter variant produces a truncated protein lacking the Sin3-like domain

  • Standard antibodies may not distinguish between full-length and truncated variants

  • Consider epitope mapping and custom antibodies targeting specific domains

• Cross-Reactivity Issues:

  • Validate antibody specificity using tissues from Zswim6 knockout models

  • Perform peptide competition assays to confirm binding specificity

• Activity-Dependent Modifications:

  • Be aware that neuronal activity states may affect ZSWIM6 localization and potentially epitope accessibility

  • Include both basal and stimulated conditions in experimental designs

How can researchers interpret conflicting ZSWIM6 localization data?

Researchers may encounter conflicting data regarding ZSWIM6 localization:

• Reconciling Nuclear vs. Cytoplasmic Observations:

  • In cultured neurons, approximately 75% of ZSWIM6 protein is found in the nucleus, with the remainder in the cytoplasm

  • Immunofluorescent staining of human cell line A-431 shows positivity primarily in the cytoplasm

  • These differences may reflect:

    • Cell type-specific localization patterns

    • Developmental stage variations

    • Functional state of the cells

• Methodological Approach to Resolve Conflicts:

  • Combine multiple detection methods (immunofluorescence, subcellular fractionation, and biochemical approaches)

  • Use tagged ZSWIM6 constructs alongside antibody detection

  • Perform super-resolution microscopy to precisely define subcellular localization

• Nuclear Localization Signal (NLS) Considerations:

  • ZSWIM6 contains an NLS sequence necessary for nuclear localization

  • When studying variants or truncated forms, assess whether the NLS is affected

  • Deletion of the NLS abolishes nuclear enrichment of ZSWIM6

• Dynamic Regulation Assessment:

  • Monitor ZSWIM6 localization under different conditions

  • Neuronal activity modulation (e.g., with picrotoxin) can decrease nuclear ZSWIM6

  • Design time-course experiments to capture dynamic changes in localization