ODF2 Antibody

Basic Research Questions

To detect ODF2 in photoreceptor cells, implement this validated protocol:

• Prepare cryo-sections of mouse retina

• Perform immunostaining using anti-ODF2 antibody (1:50-1:100 dilution)

• Co-stain with anti-γ-tubulin to confirm centrosomal localization

• Look for intense dotted staining at the outer border of the outer nuclear layer (ONL)

• Observe weak staining at the inner segments (IS) of photoreceptor cells beneath the ONL

For more detailed analysis:

• Isolate photoreceptor sensory cilium (PSC) complexes from mouse retina

• Co-stain with antibodies against acetylated tubulin and ODF2

• Identify the outer segment by detecting the primary cilium with anti-acetylated tubulin

• ODF2 colocalizes with acetylated tubulin in the outer segment and marks the connecting cilium as a dot

What are the experimental approaches to study ODF2's role in ciliopathies?

To investigate ODF2's role in ciliopathies, researchers can employ several advanced approaches:

• Tissue-specific expression analysis:

  • Examine ODF2 localization in tissues prone to ciliopathies (retina, kidney, nasal epithelium)

  • Use confocal microscopy to visualize co-localization with basal body markers

  • Look for altered expression patterns in patient samples

• Non-invasive diagnostic method development:

  • Perform immunostaining on nasal epithelial cells, which display numerous cilia anchored by basal bodies

  • This approach has been suggested as a potential non-invasive diagnostic tool for cilia-associated diseases

• Functional knockdown studies:

  • Design targeted knockdown experiments to assess functional consequences

  • Examine effects on primary cilia formation and centrosomal organization

  • Correlate phenotypes with known ciliopathies

• Mouse model analysis:

  • Study the Odf2 gene trap insertional mouse model, which shows embryonic lethality

  • Examine heterozygous mice (Odf2+/-) for partial phenotypes

  • Analyze tissue-specific effects in conditional knockout models

How does ODF2 haploinsufficiency affect sperm morphology and function?

ODF2 haploinsufficiency induces a specific type of sperm defect characterized by:

• Sperm neck-midpiece separation:

  • A new type of head-tail separation resulting in headneck sperm cells

  • Unique from other forms of decapitated sperm phenotypes

• Protein level reduction:

  • Western blotting of cauda epididymides from Odf2+/- males shows significantly reduced 75 kDa Odf2 protein compared to wild-type

  • Quantitative analysis confirms this reduction is specific to ODF2

• Specificity of defect:

  • Other tail-related proteins (Odf1, Speriolin, Tektin4, AK-1, Septin7, PHGPx, GAPDH, β-Tubulin) remain unaffected

  • This confirms that heterozygous deletion of the Odf2 gene creates a specific haploinsufficiency phenotype

This research demonstrates that even partial reduction in ODF2 levels can cause structural defects in sperm, highlighting its critical role in sperm tail integrity and male fertility.

What are the optimal methods for detecting endogenous ODF2 in centrosomes of somatic cells?

For detecting endogenous ODF2 in centrosomes:

• Cell preparation:

  • Culture cells (e.g., NIH3T3) on coverslips in appropriate media

  • Fix cells with formaldehyde (4% recommended)

  • Perform heat-mediated antigen retrieval if necessary

• Antibody selection and validation:

  • Choose antibodies validated for centrosomal localization

  • Polyclonal antibodies targeting the C-terminal region detect all known Odf2 isoforms

  • Optimal dilution for immunofluorescence: 1:10-1:100

• Co-localization strategy:

  • Always perform co-staining with anti-γ-tubulin (clone GTU-88) as a centrosomal marker

  • Use confocal microscopy to verify precise co-localization

  • Look for punctate pattern at the nuclear periphery

• Controls and validation:

  • Include negative controls (primary antibody omission)

  • Verify centrosomal specificity through siRNA knockdown of ODF2

  • Positive controls should show green dots (ODF2) overlapping with red dots (γ-tubulin)

This approach yields high-specificity detection with minimal background staining, allowing reliable identification of ODF2 at centrosomes.

How can researchers distinguish between different ODF2 isoforms?

ODF2 exists in multiple isoforms (MW 67-100 kDa), including cenexin 1 and cenexin 2, making isoform-specific detection important for comprehensive studies:

• Antibody selection strategy:

  • Use epitope-specific antibodies targeting unique regions of specific isoforms

  • For example, antibodies targeting AA 706-804 versus AA 630-829 regions may detect different isoforms

  • C-terminal region antibodies can detect all known isoforms

• Western blot optimization:

  • Use 10% SDS-PAGE for optimal separation of different molecular weight isoforms

  • Expected molecular weight range: 67-100 kDa

  • Calculated molecular weight: 89 kDa (763 amino acids)

• Tissue-specific expression analysis:

  • Different tissues may express distinct ODF2 isoforms

  • Run parallel Western blots from different tissues (testis, brain, kidney)

  • Compare band patterns and intensities to identify tissue-specific isoform expression

• Advanced techniques:

  • Use 2D gel electrophoresis to separate isoforms by both molecular weight and isoelectric point

  • Employ mass spectrometry for precise identification of specific isoforms

  • Combine with immunoprecipitation to enrich for particular isoforms

What experimental approaches can be used to study ODF2's role in primary cilia formation?

To investigate ODF2's critical function in primary cilia formation:

• Cell culture systems:

  • Generate stable cell lines with inducible ODF2 knockdown/knockout

  • Employ CRISPR-Cas9 genome editing to create precise ODF2 mutations

  • Use serum starvation to induce ciliogenesis and assess ODF2's role

• Imaging approaches:

  • Perform super-resolution microscopy to precisely localize ODF2 at the basal body

  • Use time-lapse imaging with fluorescently tagged ODF2 to track dynamic localization during ciliogenesis

  • Implement transmission electron microscopy to examine ultrastructural changes in ODF2-deficient cells

• Functional assays:

  • Assess ciliary length, frequency, and morphology in ODF2-manipulated cells

  • Examine ciliary signaling pathways (Hedgehog, Wnt) to determine functional consequences

  • Perform rescue experiments with specific ODF2 domains to identify critical regions

• Tissue-specific analysis:

  • Study renal tubular epithelial cells, which depend on primary cilia for flow sensing

  • Examine retinal photoreceptors, where ODF2 localizes to connecting cilium

  • Analyze nasal epithelial cells, which display numerous motile cilia anchored by basal bodies

These approaches can help elucidate the molecular mechanisms by which ODF2 contributes to cilia formation and maintenance, with implications for understanding ciliopathies.

What methods can be used to assess the interaction between ODF2 and other centrosomal/basal body proteins?

To characterize ODF2's interactions with other centrosomal proteins:

• Co-immunoprecipitation approaches:

  • Use anti-ODF2 antibodies (0.5-4.0 μg for 1.0-3.0 mg of total protein lysate)

  • Analyze precipitates for known centrosomal partners (CETN1, PLK1, NIN)

  • Perform reverse co-IPs to confirm interactions

• Proximity labeling techniques:

  • Generate BioID or TurboID fusions with ODF2

  • Identify proximal proteins through streptavidin pulldown and mass spectrometry

  • Validate candidates using conventional interaction assays

• Advanced microscopy:

  • Implement Förster Resonance Energy Transfer (FRET) to detect direct protein interactions

  • Use Proximity Ligation Assay (PLA) to visualize protein interactions in situ

  • Apply Structured Illumination Microscopy (SIM) or STORM to resolve spatial relationships at the centrosome

• Functional interaction studies:

  • Perform depletion-rescue experiments targeting specific interaction domains

  • Assess centrosome/basal body assembly in the absence of specific interactions

  • Engineer mutations in predicted interaction surfaces to disrupt specific protein pairs

• In vitro binding assays:

  • Use recombinant ODF2 domains to perform direct binding studies

  • Map interaction domains through truncation analysis

  • Quantify binding affinities using biophysical techniques (SPR, ITC)

These methodological approaches will help elucidate ODF2's role as a scaffold protein at the centrosome and basal body, providing insights into its functions in centrosome maturation and ciliogenesis.

What are the optimal fixation and permeabilization conditions for ODF2 immunostaining?

Based on validated protocols, optimal conditions for ODF2 immunostaining include:

• Fixation options:

  • Formaldehyde fixation (4%) is recommended for most applications

  • For retinal tissue sections, cryofixation followed by sectioning yields good results

• Antigen retrieval:

  • For IHC applications, heat-mediated antigen retrieval is recommended

  • Primary option: TE buffer pH 9.0

  • Alternative option: citrate buffer pH 6.0

• Permeabilization:

  • For cultured cells: 0.1-0.5% Triton X-100 in PBS for 5-10 minutes

  • For tissue sections: longer permeabilization may be required (15-30 minutes)

  • For centrosomal structures, gentle permeabilization is crucial to preserve structure

• Blocking conditions:

  • 5% normal serum (from secondary antibody host species)

  • 1-3% BSA in PBS

  • Include 0.1% Triton X-100 in blocking solution for continued permeabilization

These conditions help preserve antigenicity while allowing antibody access to subcellular structures, resulting in optimal signal-to-noise ratio for ODF2 detection.

How can researchers troubleshoot weak or non-specific staining with ODF2 antibodies?

When encountering staining issues with ODF2 antibodies, implement this systematic troubleshooting approach:

• For weak or no signal:

  • Verify antibody reactivity with your species (human, mouse, rat are validated)

  • Increase antibody concentration (try 1:10 dilution for IF/ICC)

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

  • Try different antigen retrieval methods (TE buffer pH 9.0 or citrate buffer pH 6.0)

  • Check tissue/sample preparation (fresh samples yield better results)

  • Use signal amplification systems (tyramide signal amplification)

• For high background or non-specific staining:

  • Increase blocking time and concentration (try 5% BSA)

  • Add 0.1-0.3% Triton X-100 to reduce non-specific binding

  • Reduce primary antibody concentration

  • Include additional washing steps (5x 5 minutes)

  • Pre-adsorb antibody with non-specific proteins

  • Include controls (primary antibody omission, isotype control)

• For centrosomal/basal body detection:

  • Always co-stain with established markers (γ-tubulin for centrosomes)

  • Use confocal microscopy for better resolution of small structures

  • Consider super-resolution microscopy for detailed localization studies

  • Ensure cells/tissues are in appropriate cell cycle stage (G1/G0 for primary cilia)

• Validation controls:

  • Include known positive tissue controls (mouse testis, mouse lung)

  • Use ODF2 knockdown/knockout samples as negative controls

  • Verify antibody specificity by Western blot before immunostaining

What storage and handling practices ensure optimal ODF2 antibody performance?

To maintain antibody functionality and extend shelf-life:

• Storage recommendations:

  • Store at -20°C as recommended by manufacturers

  • Antibodies are typically stable for one year after shipment when properly stored

  • For conjugated antibodies (e.g., fluorescent dyes), avoid exposure to light

• Buffer composition:

  • Most ODF2 antibodies are supplied in PBS with stabilizers:

    • Typically contains 0.02% sodium azide and 50% glycerol pH 7.3

    • Some formulations include 0.05% Proclin300 and 0.5% BSA

    • Small volume formats (20μl) may contain 0.1% BSA

• Aliquoting considerations:

  • For large volume antibodies, create single-use aliquots to avoid freeze-thaw cycles

  • For -20°C storage, aliquoting is often unnecessary as stated by manufacturers

  • If aliquoting, use sterile tubes and aseptic technique

• Working solutions:

  • Dilute only the amount needed for immediate use

  • Prepare fresh working solutions for each experiment

  • Store diluted antibody at 4°C for short-term use (1-2 weeks maximum)

  • Add BSA (0.5-1%) to diluted antibody to improve stability