spata45 Antibody

What is SPATA45 and why is it relevant to reproductive biology research?

SPATA45 (Spermatogenesis Associated Protein 45) is a protein encoded by the C1ORF227 gene in humans. While specific functional studies on SPATA45 are still emerging, it belongs to the SPATA family of proteins which are typically involved in spermatogenesis and reproductive processes. Current evidence suggests it may play roles in sperm development or function. Research using antibodies against SPATA45 helps elucidate its localization patterns, expression levels, and potential protein interactions in reproductive tissues. Unlike better-characterized family members like SPAG5 (which is known to be necessary for normal spindle formation during mitosis), SPATA45's precise functions require further investigation using validated antibodies for proper characterization .

How do I validate the specificity of a SPATA45 antibody before experimental use?

Antibody validation is critical for ensuring experimental reliability. For SPATA45 antibodies, a multi-step validation approach is recommended:

• Blocking peptide competition assay: Use a recombinant SPATA45 protein antigen such as the one available with N-terminal His6-ABP tag corresponding to human C1ORF227 as a blocking peptide. Pre-incubate your antibody with excess blocking peptide before application in your experiment. Disappearance of signal confirms specificity .

• Western blot analysis: Run parallel samples with and without SPATA45 knockdown/knockout. Compare the molecular weight of detected bands (expected ~27kDa for SPATA45) and ensure signal reduction in knockdown samples.

• Cross-reactivity testing: Test the antibody on tissues/cells known to express or not express SPATA45 based on RNA-seq data.

• Orthogonal method verification: Compare protein detection results with mRNA expression data from qPCR or RNA-seq.

The recombinant protein antigen purified by IMAC chromatography (>80% purity) is specifically designed for antibody validation purposes and yields reliable results in competition assays .

What are the key differences between polyclonal and monoclonal antibodies against SPATA45?

The choice between polyclonal and monoclonal should be guided by experimental goals. For reproductive biology studies examining SPATA45 expression patterns, polyclonal antibodies often provide better sensitivity for detection in diverse tissues .

What are the optimal conditions for using SPATA45 antibodies in Western blot analysis?

Successful Western blot detection of SPATA45 requires careful optimization of conditions:

• Sample preparation:

  • Tissues: Homogenize reproductive tissues in RIPA buffer supplemented with protease inhibitors

  • Cell lines: Lyse in buffer containing 50mM Tris-HCl pH 7.4, 150mM NaCl, 1% NP-40, 0.5% sodium deoxycholate with protease inhibitors

  • Include phosphatase inhibitors if studying phosphorylation states

• Gel conditions:

  • Use 12-15% SDS-PAGE gels to resolve the ~27kDa SPATA45 protein effectively

  • Load 20-50μg of total protein per lane

• Transfer conditions:

  • Semi-dry or wet transfer at 100V for 1 hour using PVDF membranes (preferred over nitrocellulose for this protein)

• Blocking:

  • 5% non-fat dry milk in TBST for 1 hour at room temperature

• Primary antibody incubation:

  • Dilution: 1:500-1:2000 (similar to SPAG5 antibody recommendations)

  • Incubate overnight at 4°C in 5% BSA in TBST

• Detection:

  • Use HRP-conjugated secondary antibodies and enhance detection with extended exposure times if signal is weak

  • Expected band: 27kDa (may appear slightly higher due to post-translational modifications)

• Controls:

  • Include blocking peptide competition control using SPATA45 recombinant protein

  • Run testis tissue extracts as positive control

How should I optimize immunohistochemistry protocols for SPATA45 detection in reproductive tissues?

For optimal SPATA45 detection in reproductive tissues by IHC:

• Tissue preparation:

  • Fixation: 4% paraformaldehyde or 10% neutral buffered formalin (12-24 hours)

  • Processing: Standard paraffin embedding

  • Sectioning: 4-5μm thickness

• Antigen retrieval:

  • Heat-mediated retrieval using TE buffer pH 9.0 (similar to SPAG5 protocol)

  • Alternative: Citrate buffer pH 6.0 (test both to determine optimal conditions for your specific SPATA45 antibody)

  • Heating time: 20 minutes at 95-98°C

• Blocking and permeabilization:

  • Block endogenous peroxidase: 3% H₂O₂ for 10 minutes

  • Permeabilize: 0.2% Triton X-100 in PBS for 10 minutes

  • Block non-specific binding: 5% normal serum (species of secondary antibody) with 1% BSA

• Antibody incubation:

  • Primary antibody dilution: 1:50-1:500 based on antibody specificity

  • Incubation: Overnight at 4°C in humidified chamber

  • Secondary antibody: HRP-polymers or biotinylated secondaries

• Visualization:

  • DAB substrate for brown staining

  • Counterstain: Hematoxylin (light)

• Controls:

  • Positive control: Human or mouse testis tissue

  • Negative control: Primary antibody omission

  • Blocking peptide competition using SPATA45 recombinant protein

What approaches can be used to study SPATA45 protein-protein interactions?

To investigate SPATA45's interacting partners in reproductive biology:

• Co-immunoprecipitation:

  • Use 0.5-4.0μg antibody per 1.0-3.0mg total protein lysate (similar to SPAG5 antibody recommendations)

  • Cross-link antibody to protein A/G beads to prevent heavy chain interference

  • Use gentler lysis buffers (150mM NaCl, 10mM Tris pH 7.4, 1mM EDTA, 1% NP-40) to preserve protein complexes

  • Include appropriate negative controls (IgG from same species as SPATA45 antibody)

• Proximity ligation assay (PLA):

  • Combine SPATA45 antibody with antibodies against suspected interacting partners

  • Useful for detecting interactions in situ in tissue sections

  • Quantify fluorescent spots to assess interaction frequency

• FRET/BRET approaches:

  • Generate fluorescent protein fusions with SPATA45 and potential partners

  • Measure energy transfer as indication of physical proximity

• Yeast two-hybrid screening:

  • Use SPATA45 as bait to screen testis cDNA libraries

  • Validate hits using reciprocal co-IP with SPATA45 antibodies

• Mass spectrometry following IP:

  • Immunoprecipitate SPATA45 using validated antibodies

  • Identify co-precipitating proteins through LC-MS/MS

  • Verify interactions through reciprocal IP

How do I address non-specific binding when using SPATA45 antibodies?

Non-specific binding is a common challenge with antibodies to less-characterized proteins like SPATA45. Implement these strategies to minimize background:

• Antibody validation:

  • Always perform blocking peptide competition assays with SPATA45 recombinant protein

  • The recombinant protein should have >80% purity and be formulated in PBS with 1M Urea, pH 7.4

• Blocking optimization:

  • Test different blocking agents: 5% BSA, 5% normal serum, commercial blocking buffers

  • Extend blocking time to 2 hours at room temperature

  • Add 0.05-0.1% Tween-20 to reduce hydrophobic interactions

• Antibody dilution:

  • Titrate antibody to find minimal effective concentration

  • Prepare in fresh blocking buffer

  • Consider overnight incubation at 4°C instead of shorter times at room temperature

• Washing protocols:

  • Increase wash duration and number of washes (5 x 5 minutes)

  • Use PBS-T with 0.1% Tween-20 for more stringent washing

• Cross-adsorption:

  • Pre-adsorb antibody with tissue/cell lysates from organisms not expressing the target

  • Remove cross-reacting antibodies by incubating with immobilized proteins from non-relevant tissues

• Reducing auto-fluorescence:

  • For fluorescent detection, treat sections with Sudan Black B (0.1% in 70% ethanol)

  • Use specific quenching reagents like TrueBlack or commercial autofluorescence quenchers

What are the best storage conditions to maintain SPATA45 antibody performance over time?

Proper storage is crucial for maintaining antibody performance:

• Temperature conditions:

  • Store antibody aliquots at -20°C for long-term storage

  • Avoid repeated freeze-thaw cycles (limit to <5)

  • For working solutions, store at 4°C for up to 2 weeks with preservative

• Buffer considerations:

  • Optimal storage buffer: PBS with 0.02% sodium azide and 50% glycerol, pH 7.3

  • Glycerol prevents freeze-thaw damage

  • Sodium azide prevents microbial contamination

• Aliquoting strategy:

  • Prepare small single-use aliquots (10-20μL)

  • Use sterile microcentrifuge tubes

  • Record date of aliquoting and track usage

• Performance monitoring:

  • Test antibody performance periodically on positive control samples

  • Document lot-to-lot variations

  • Consider including BSA (0.1%) for antibodies at lower concentrations

• Shipping/temporary storage:

  • Use ice packs for short-term transport

  • Avoid exposure to heat or direct sunlight

  • Return to -20°C as soon as possible

How can I determine if my SPATA45 antibody has undergone degradation or denaturation?

Monitor antibody quality using these approaches:

• Performance assays:

  • Compare current results with historical positive controls

  • Declining signal intensity or increasing background suggests degradation

  • Test on known positive samples (e.g., testis tissue) at regular intervals

• Physical inspection:

  • Check for visible precipitates, cloudiness, or color changes

  • Centrifuge at 10,000g for 5 minutes to identify aggregation

  • Mild precipitates can sometimes be resolubilized at room temperature

• Analytical methods:

  • SDS-PAGE to check for fragmentation patterns

  • Size exclusion chromatography to assess aggregation state

  • ELISA against original immunogen to quantify binding capacity

• Functional verification:

  • Western blot for expected band at 27kDa

  • Competition assay with SPATA45 recombinant protein

  • Signal-to-noise ratio comparison with fresh antibody

• Regeneration approaches:

  • Centrifugation to remove aggregates (10,000g, 10 minutes)

  • Filtration through 0.22μm filters

  • Buffer exchange via dialysis or desalting columns

  • Note: severely degraded antibodies cannot be rescued

How can SPATA45 antibodies be applied in studying spermatogenesis defects and male infertility?

SPATA45 antibodies provide valuable tools for investigating reproductive biology:

• Expression profiling across spermatogenesis stages:

  • Use immunohistochemistry on testis sections to map SPATA45 expression in:

    • Spermatogonia

    • Primary/secondary spermatocytes

    • Round/elongating spermatids

    • Mature spermatozoa

  • Compare expression patterns between normal and pathological samples

• Correlation analysis with fertility parameters:

  • Quantify SPATA45 levels in sperm samples from men with different fertility status

  • Correlate with sperm count, motility, morphology, and fertilization rates

  • Implement computer-assisted image analysis for objective quantification

• Genetic model systems:

  • Generate SPATA45 knockout/knockdown models

  • Use antibodies to confirm protein depletion

  • Assess reproductive phenotypes and developmental defects

• Mechanistic studies:

  • Investigate SPATA45 phosphorylation states during sperm capacitation

  • Examine protein-protein interactions during acrosome reaction

  • Study subcellular localization changes during fertilization

• Clinical applications:

  • Develop diagnostic immunoassays for SPATA45 in seminal plasma

  • Evaluate SPATA45 as a biomarker for specific types of male infertility

  • Assess correlation with success rates in assisted reproductive technologies

What protocols exist for using SPATA45 antibodies in high-resolution imaging techniques?

Advanced imaging applications for SPATA45 include:

• Super-resolution microscopy:

  • STORM/PALM: Use photoconvertible fluorophore-conjugated secondary antibodies

  • SIM: Standard immunofluorescence protocol with high-quality primary SPATA45 antibody

  • Sample preparation: Thinner sections (2-3μm) and longer antibody incubation times

  • Fixation: 4% PFA followed by moderate permeabilization (0.1% Triton X-100)

• Expansion microscopy:

  • Pre-expansion immunolabeling: Apply SPATA45 antibody before hydrogel embedding

  • Post-expansion immunolabeling: Apply antibody after expansion

  • Anchor antibodies with NHS-ester chemistry to prevent dissociation

  • Expansion factor: 4-5x linear expansion for reproductive tissues

• Electron microscopy:

  • Immunogold labeling: Use 5-15nm gold-conjugated secondary antibodies

  • Sample preparation: LR White or Lowicryl embedding for optimal antigenicity

  • Antibody dilutions: 2-5x more concentrated than for light microscopy

  • Post-embedding vs. pre-embedding protocols based on epitope accessibility

• Correlative light and electron microscopy (CLEM):

  • Immunofluorescence with SPATA45 antibody on thin sections

  • Transfer to EM grid and perform gold labeling

  • Use fiducial markers for correlation between modalities

• Live-cell imaging considerations:

  • Develop SPATA45-specific nanobodies for live applications

  • Use cell-permeable antibody fragments

  • Validate specificity with fixed-cell imaging using conventional antibodies

How can computational approaches enhance data interpretation from SPATA45 antibody-based experiments?

Integrate computational methods with antibody-based detection:

• Quantitative image analysis:

  • Automated detection of SPATA45-positive cells in tissue sections

  • Machine learning algorithms for pattern recognition in subcellular localization

  • Colocalization analysis with organelle markers (Pearson's correlation, Manders' overlap)

  • 3D reconstruction from confocal z-stacks to assess spatial distribution

• Multi-omics data integration:

  • Correlate SPATA45 protein levels (immunoblotting) with transcriptomics data

  • Network analysis to identify functional protein clusters

  • Pathway enrichment analysis of SPATA45-interacting proteins

• Structural prediction and epitope mapping:

  • In silico prediction of SPATA45 structure

  • Identification of antibody binding sites through peptide arrays

  • Molecular dynamics simulations to predict conformational epitopes

• Temporal dynamics analysis:

  • Time-lapse imaging quantification of SPATA45 during cell cycle phases

  • Signal intensity tracking across developmental stages

  • Mathematical modeling of expression patterns during spermatogenesis

• Population heterogeneity assessment:

  • Single-cell analysis of SPATA45 levels in complex tissues

  • Cluster analysis to identify cell subpopulations

  • Pseudo-time analysis for developmental trajectories

How does SPATA45 expression compare with other SPATA family members during gametogenesis?

Comparative analysis of SPATA family proteins:

Methodological approaches for comparative studies:

• Multiplex immunofluorescence:

  • Use antibodies raised in different species

  • Employ tyramide signal amplification for sequential detection

  • Carefully validate antibody combinations to prevent cross-talk

• Developmental timing analysis:

  • Compare expression across precisely staged samples

  • Use age-matched tissues for temporal profiles

  • Align with established markers of spermatogenic stages

• Functional redundancy testing:

  • Knockdown studies with rescue experiments

  • Investigate compensatory changes in other family members

  • Assess overlapping binding partners

What are the considerations for using SPATA45 antibodies in flow cytometry and cell sorting?

Flow cytometry optimization for SPATA45 detection:

• Cell preparation:

  • Single-cell suspensions from testicular tissue:

    • Enzymatic digestion (collagenase IV + DNase I)

    • Gentle mechanical dissociation

    • Filtration through 40μm cell strainers

  • Fixation options:

    • 2% PFA for 10 minutes (maintains structure)

    • 70% ethanol for intracellular epitopes

    • Commercial fixation buffers optimized for nuclear proteins

• Permeabilization protocols:

  • Saponin (0.1%) for cytoplasmic epitopes

  • Triton X-100 (0.1%) for nuclear epitopes

  • Commercial permeabilization buffers with protein stabilizers

• Antibody titration:

  • Test multiple concentrations (typically 0.1-10μg/mL)

  • Evaluate signal-to-noise ratio using positive and negative populations

  • Include blocking peptide competition controls

• Multi-parameter panel design:

  • Combine with DNA content staining (DAPI, Hoechst)

  • Add stage-specific markers (PLZF, SCP3, PNA)

  • Include viability dye to exclude dead cells

• Sorting considerations:

  • Use higher antibody concentrations to improve detection

  • Sort buffer optimization to maintain viability

  • Post-sort validation by immunofluorescence or RT-PCR

• Analysis approaches:

  • Histogram overlays to compare expression levels

  • Bivariate plots with matched isotype controls

  • Quantification of positive cell percentages across experimental conditions

How can somatic hypermutation principles be applied to generating high-affinity SPATA45 antibodies for research?

Leveraging antibody diversity mechanisms to improve SPATA45 antibody quality:

• Phage display technology:

  • Create antibody fragment (Fab, scFv) libraries

  • Perform multiple rounds of selection against SPATA45 protein

  • Introduce random mutations to mimic somatic hypermutation

  • Screen for affinity improvements using competition ELISA

• Antibody engineering approaches:

  • CDR grafting and framework optimization

  • Structure-guided mutagenesis of antibody paratopes

  • Affinity maturation through directed evolution

  • Humanization for therapeutic applications

• Natural process exploitation:

  • Sequential immunization strategies with different SPATA45 epitopes

  • Extended immunization protocols to allow natural affinity maturation

  • Isolation of B-cells from immunized animals for antibody gene cloning

• Hybridoma optimization:

  • Subcloning of hybridoma cells to select high-producers

  • CRISPR-based engineering of hybridoma cell lines

  • Culture condition optimization for improved antibody yield

• Quality assessment metrics:

  • Surface plasmon resonance for affinity measurement

  • Epitope binning to identify diverse binding sites

  • Cross-reactivity profiling against related SPATA family members

The principles of somatic hypermutation, which naturally increase antibody affinity and specificity during immune responses, can be harnessed through biotechnological approaches to develop superior research antibodies against challenging targets like SPATA45 .

What emerging techniques might enhance SPATA45 antibody applications in reproductive biology research?

The field of SPATA45 research will benefit from several emerging technologies:

• Spatial transcriptomics integration:

  • Combining antibody detection with spatial RNA sequencing

  • Correlating protein localization with gene expression patterns

  • Single-cell resolution mapping of SPATA45 in intact tissues

• Engineered antibody fragments:

  • Single-domain antibodies (nanobodies) for improved tissue penetration

  • Bispecific formats to simultaneously detect SPATA45 and interacting partners

  • Cell-permeable antibody fragments for live-cell applications

• CRISPR knock-in approaches:

  • Endogenous tagging of SPATA45 to overcome antibody limitations

  • Validation of antibody specificity through targeted epitope modification

  • Creation of reporter systems for dynamic expression monitoring

• Mass cytometry (CyTOF):

  • Metal-tagged antibodies for highly multiplexed detection

  • Simultaneous assessment of dozens of markers alongside SPATA45

  • Improved quantification through absence of spectral overlap

• Antibody-based proximity labeling:

  • APEX2 or BioID fusion with anti-SPATA45 antibodies

  • Mapping spatial interactomes in specific cellular compartments

  • Temporal control of labeling for dynamic interaction studies

These approaches will provide unprecedented insights into SPATA45 biology in reproductive processes and potentially reveal new therapeutic targets for reproductive disorders .