PMFBP1 Antibody

What is PMFBP1 and what is its role in spermatogenesis?

PMFBP1 (polyamine modulated factor 1 binding protein 1) is a testis-specific protein with a canonical length of 1007 amino acid residues and a mass of 117.5 kDa in humans. Its subcellular localization is primarily in cell projections, particularly at the head-tail junction of spermatozoa. PMFBP1 is essential for normal spermatogenesis, forming a critical "sandwich" structure with SUN5 and SPATA6 proteins .

PMFBP1 functions as a scaffold protein connecting the coupling device and the sperm nuclear membrane. Research indicates that it sits between SUN5 (a nuclear membrane protein) and SPATA6 (a structural protein), where it plays a vital role in maintaining the integrity of the head-tail junction in sperm . Mutations in the PMFBP1 gene are strongly associated with acephalic spermatozoa syndrome (ASS), a condition characterized by sperm heads detaching from tails, leading to male infertility .

What applications are PMFBP1 antibodies commonly used for?

PMFBP1 antibodies are utilized in multiple research applications including:

When selecting an antibody, researchers should consider the specific application requirements and validated reactivity for their experimental model .

What species reactivity do commercial PMFBP1 antibodies exhibit?

Most commercially available PMFBP1 antibodies demonstrate reactivity against human samples, with some also showing cross-reactivity with mouse models . According to the search results, PMFBP1 gene orthologs have been reported in mouse, rat, bovine, frog, zebrafish, chimpanzee, and chicken species .

When selecting antibodies, it's important to verify the validated species reactivity:

Always perform validation tests when using these antibodies in species not explicitly listed in manufacturer specifications.

How should I optimize Western blot protocols for PMFBP1 detection?

For optimal Western blot detection of PMFBP1, consider the following protocol recommendations:

• Sample preparation:

  • For testicular tissue: Homogenize in RIPA buffer with protease inhibitor cocktail

  • For sperm samples: Use specialized lysis buffers such as TAP lysis buffer (50 mM HEPES-KOH [pH 7.5], 100 mM KCl, 2 mM EDTA, 10% glycerol, 0.1% NP-40, 10 mM NaF, 0.25 mM Na₃VO₄, 50 mM β-glycerolphosphate) plus protease inhibitors

• Protein loading and separation:

  • Use 8-10% SDS-PAGE gels to properly resolve the 117-119 kDa PMFBP1 protein

  • Load adequate protein (40-50 μg) for detection of endogenous PMFBP1

• Antibody incubation:

  • Primary antibody: Dilute rabbit polyclonal anti-PMFBP1 antibody at 1:500-1:2000 in blocking buffer and incubate overnight at 4°C

  • Secondary antibody: Use HRP-conjugated goat anti-rabbit IgG (typically 1:10,000 dilution)

• Controls:

  • Positive control: Use mouse testis tissue, HepG2 cells, or MCF-7 cells

  • Negative control: Use tissues with no known PMFBP1 expression

  • Loading control: Anti-GAPDH antibody (1:10,000) is commonly used

When troubleshooting, note that PMFBP1 typically appears as a specific 117-kDa band on Western blots. If studying mutant PMFBP1 proteins, expect potentially reduced band intensity compared to wild-type controls .

What is the optimal protocol for immunofluorescence detection of PMFBP1 in sperm samples?

For immunofluorescence detection of PMFBP1 in sperm samples, follow this optimized protocol:

• Sample preparation:

  • Spread spermatozoa on glass slides and allow to air dry

  • Fix in 4% paraformaldehyde at room temperature for 15-20 minutes

  • Wash slides with PBS three times

• Blocking and permeabilization:

  • Block with 5% bovine serum albumin for 1 hour at room temperature

  • For sperm cells, add 0.1% Triton X-100 to permeabilize membranes

• Antibody incubation:

  • Primary antibody: Dilute PMFBP1 antibody (0.25-2 μg/mL) and incubate overnight at 4°C

  • Secondary antibody: Use fluorophore-conjugated secondary antibody appropriate for your imaging system

• Nuclear counterstaining and imaging:

  • Counterstain nuclei with DAPI

  • For optimal results, image immediately using confocal microscopy (e.g., LSM 780/710 or SP8 microscope)

• Controls and co-staining:

  • Include nuclear membrane markers (e.g., SUN5) and acrosomal markers (e.g., CD46 or sp56) for colocalization studies

  • Use spermatozoa from known PMFBP1 mutation carriers as comparison when studying pathogenic variants

In normal sperm, PMFBP1 should appear localized to the head-tail junction. In acephalic spermatozoa syndrome cases, the signal may be weaker or mislocalized .

How can I design experiments to investigate PMFBP1 mutations and their effects on protein expression?

To investigate PMFBP1 mutations and their effects on protein expression, consider this experimental approach:

• Mutation identification and validation:

  • Screen patient samples with acephalic spermatozoa syndrome using Sanger sequencing of PMFBP1 coding regions

  • Perform whole exome sequencing (WES) for comprehensive mutation identification

  • Verify mutations using in silico prediction tools (SIFT, Polyphen2, Mutation Taster)

• Expression analysis in patient samples:

  • Collect sperm samples from patients with identified PMFBP1 mutations

  • Perform Western blot analysis to quantify PMFBP1 expression levels

  • Compare band intensity between patient samples and normal controls

• In vitro mutation modeling:

  • Generate plasmid constructs containing wild-type or mutant PMFBP1 sequences

  • Transfect cells (e.g., HeLa, HEK293T) with these constructs

  • Assess protein expression by Western blot and localization by immunofluorescence

• Protein degradation pathway analysis:

  • Treat cells expressing mutant PMFBP1 with proteasome inhibitors (e.g., MG132)

  • Analyze whether inhibitor treatment restores mutant protein expression

  • This approach can confirm if mutant PMFBP1 undergoes degradation via the ubiquitination pathway

• Predicting effects of mutations:

  • Use Open Reading Frame Finder (https://www.ncbi.nlm.nih.gov/orffinder/) to predict new ORFs from mutated PMFBP1 mRNA

  • Assess conservation of affected amino acids across species

This comprehensive approach allows robust characterization of how specific mutations affect PMFBP1 expression, stability, and function.

What methodological approaches can be used to study protein-protein interactions involving PMFBP1?

To investigate protein-protein interactions involving PMFBP1, particularly with SUN5 and SPATA6 in the head-tail coupling apparatus, employ these experimental approaches:

• Co-immunoprecipitation (Co-IP):

  • Clone SUN5 and SPATA6 into pRK vector with FLAG tag

  • Clone PMFBP1 into pEGFP-C1 vector

  • Co-transfect constructs into HEK293T cells

  • Lyse cells in TAP lysis buffer (50 mM HEPES-KOH [pH 7.5], 100 mM KCl, 2 mM EDTA, 10% glycerol, 0.1% NP-40, 10 mM NaF, 0.25 mM Na₃VO₄, 50 mM β-glycerolphosphate) with protease inhibitors

  • Immunoprecipitate with anti-FLAG antibody overnight at 4°C

  • Incubate with protein A-Sepharose for 2 hours at 4°C

  • Wash precipitants with IP buffer

  • Analyze by Western blot using appropriate antibodies

• GST pull-down assays:

  • Express PMFBP1 as a GST fusion protein

  • Express potential interacting partners with different tags

  • Perform pull-down experiments to confirm direct interactions

  • Analyze using SDS-PAGE and Western blotting

• Proximity ligation assay (PLA):

  • Use fixed sperm or transfected cells

  • Apply antibodies against PMFBP1 and potential interacting partners

  • Perform PLA according to manufacturer's protocol

  • This technique allows visualization of protein interactions in situ

• Immunofluorescence co-localization:

  • Perform double immunofluorescence staining for PMFBP1 and interacting partners

  • Use confocal microscopy to analyze co-localization patterns

  • Calculate Pearson's correlation coefficient to quantify co-localization

• Functional rescue experiments:

  • Expose spermatogenic cells to exogenous recombinant PMFBP1 protein (e.g., from Met1 to Lys120 of N-terminus, 20 ng/ml)

  • Analyze changes in expression of interacting partners (ODF1, ODF2)

  • Compare with control cells exposed to random protein fragments

These complementary approaches provide robust evidence for specific protein-protein interactions involving PMFBP1.

How can I design CRISPR/Cas9 knockout models to study PMFBP1 function?

To generate and analyze PMFBP1 knockout models using CRISPR/Cas9 technology, follow these methodological guidelines:

• gRNA design and validation:

  • Design guide RNAs targeting conserved exons of PMFBP1

  • Validate gRNA efficiency using in vitro cleavage assays

  • Consider targeting early exons to ensure complete protein disruption

• Generation of knockout mice:

  • Inject Cas9 protein and validated gRNAs into mouse zygotes

  • Transfer embryos to pseudopregnant females

  • Screen offspring for PMFBP1 mutations using PCR and sequencing

• Genotyping strategy:

  • Design primers specific for wild-type and knockout alleles

  • Example primers from previous studies:

    • For wild-type allele: Forward 5′-GATGAGTAATAACAGCCCAGG-3′ and Reverse 5′-CTTGATGCATGCGCAGTTAG-3′

    • For Pmfbp1 knockout allele: Forward 5′-GATCTCAGACTCCCTCGTCAACTC-3′ and Reverse 5′-GGAGTGGGCAGGATTGAAAG-3′

• Phenotypic characterization:

  • Assess fertility through breeding tests

  • Analyze sperm parameters (count, motility, morphology)

  • Examine sperm structure using light and electron microscopy

  • Perform histological analysis of testicular tissue

• Molecular characterization:

  • Analyze protein expression by Western blot

  • Determine subcellular localization using immunofluorescence

  • Examine expression of interacting partners (SUN5, SPATA6)

  • Investigate potential compensatory mechanisms

• Functional studies:

  • Perform in vitro fertilization assays to assess sperm function

  • Examine acrosome reaction and capacitation in knockout sperm

  • Analyze head-tail coupling using specialized staining techniques

Remember to conduct all animal experiments in accordance with institutional animal care protocols. Previous studies have used protocols like #08-133 from the Institute of Zoology, Chinese Academy of Sciences .

How can I troubleshoot weak or absent signals when using PMFBP1 antibodies?

When encountering weak or absent signals with PMFBP1 antibodies, systematically address these potential issues:

• Sample quality and preparation issues:

  • Ensure protein degradation hasn't occurred by adding fresh protease inhibitors

  • For sperm samples, verify that cells are properly lysed using specialized buffers

  • Check protein quantification method accuracy

  • For immunohistochemistry, ensure proper fixation and antigen retrieval

• Antibody selection and optimization:

  • Verify antibody reactivity matches your species of interest

  • Test different antibody concentrations (recommended range: 0.25-2 μg/mL for IF; 1:500-1:2000 for WB)

  • Try alternative PMFBP1 antibodies targeting different epitopes:

    • Mouse monoclonal (e.g., Invitrogen MA5-24491)

    • Rabbit polyclonal (e.g., Proteintech 17061-1-AP, Invitrogen PA5-69495)

• Detection system troubleshooting:

  • Use fresh secondary antibodies at appropriate dilutions

  • For fluorescence applications, minimize photobleaching

  • For HRP-based detection, prepare fresh substrate solution

• Biological considerations:

  • Remember PMFBP1 is tissue-specific with highest expression in testis

  • Expression may be significantly reduced in samples with PMFBP1 mutations

  • Consider that PMFBP1 mutant proteins may be degraded via ubiquitination pathway

• Positive controls:

  • Include known positive samples (mouse testis tissue, HepG2 cells, or MCF-7 cells)

  • Consider using recombinant PMFBP1 protein as a control

If troubleshooting a specific mutation study, note that some PMFBP1 missense mutations (e.g., c.301A>C; p.T101P) significantly reduce protein levels but don't eliminate expression entirely .

How can I distinguish between wild-type and mutant PMFBP1 in experimental settings?

Distinguishing between wild-type and mutant PMFBP1 requires careful experimental design:

• Western blot analysis:

  • Compare band intensity between samples

  • For missense mutations, expect reduced intensity but same molecular weight (117-119 kDa)

  • For truncating mutations, look for lower molecular weight bands or absence of signal

  • Consider using multiple antibodies targeting different epitopes

• Immunofluorescence comparison:

  • In normal samples, PMFBP1 localizes to the head-tail junction of spermatozoa

  • In mutant samples, signal intensity is typically reduced

  • Missense mutations (e.g., p.T101P) may maintain correct localization but with reduced signal

  • Some mutations may affect localization patterns

• Proteasome inhibition experiments:

  • Treat cells expressing mutant PMFBP1 with MG132 (proteasome inhibitor)

  • If signal intensity increases after treatment, this suggests the mutant protein undergoes degradation via ubiquitination

  • This approach has successfully demonstrated degradation of the p.T101P mutant

• Recombinant protein expression:

  • Express wild-type and mutant PMFBP1 in heterologous systems

  • Compare expression levels, stability, and localization

  • Analyze functional interactions with partner proteins

• PCR-based methods for genotyping:

  • Design mutation-specific primers for PCR

  • Perform allele-specific PCR to distinguish wild-type from mutant sequences

  • Confirm results with Sanger sequencing

These approaches provide complementary data to distinguish between wild-type and mutant PMFBP1 proteins in both research and clinical settings.

How can PMFBP1 antibodies be used to study male infertility pathogenesis?

PMFBP1 antibodies offer valuable tools for investigating male infertility, particularly acephalic spermatozoa syndrome:

• Clinical diagnostics:

  • Use immunofluorescence to screen sperm samples from infertile men

  • Identify patients with abnormal PMFBP1 expression or localization

  • Correlate antibody staining patterns with genetic analysis results

• Mechanistic studies:

  • Investigate PMFBP1's role in the head-tail coupling apparatus

  • Study interactions with SUN5, SPATA6, and other proteins

  • Analyze changes in these interactions in infertile patients

• Genetic correlation studies:

  • Compare PMFBP1 protein expression between patients with different mutations

  • Link specific mutation types (nonsense, missense, frameshift) to protein expression patterns

  • Create a comprehensive database of mutations and their effects on protein expression

• Therapeutic development:

  • Screen compounds that might stabilize mutant PMFBP1 proteins

  • Test exogenous PMFBP1 administration to rescue phenotypes in cellular models

  • Develop targeted approaches for patients with specific mutation types

• Comparative studies across species:

  • Use PMFBP1 antibodies to study evolutionary conservation

  • Compare expression patterns in different mammalian species

  • Investigate species-specific adaptations in the head-tail coupling mechanism

These applications contribute to understanding the molecular basis of male infertility and may eventually lead to novel diagnostic and therapeutic approaches.

What are the current limitations of PMFBP1 antibodies and how might they be addressed in future research?

Current limitations of PMFBP1 antibodies and potential solutions include:

Advanced approaches to address these limitations include:

• Development of monoclonal antibodies with defined epitope recognition

• Creation of recombinant antibody fragments with enhanced specificity

• Generation of nanobodies against PMFBP1 for improved access to structural epitopes

• Application of proximity labeling techniques to study PMFBP1 interaction networks

• Development of CRISPR knock-in models with epitope-tagged endogenous PMFBP1