dcaf17 Antibody

What is DCAF17 and what are its known biological functions?

DCAF17, also known as C2orf37, functions as a substrate receptor for CUL4-DDB1 E3 ubiquitin-protein ligase complex . This nucleolar protein exists in two isoforms: alpha (453 amino acid residues) and beta (520 amino acid residues) . DCAF17 plays critical roles in several cellular processes:

• Substrate recruitment for protein ubiquitination

• Essential role in spermatogenesis and male fertility

• Involvement in nucleolar functions including ribosome biogenesis, cell cycle regulation, cellular aging, signal recognition, RNA processing, and apoptosis

Expression analysis demonstrates highest DCAF17 levels in testis, with moderate expression in brain, liver, and pancreas. During postnatal development, testicular DCAF17 mRNA levels gradually increase with age until 32 days postpartum before plateauing .

What applications are commercially available DCAF17 antibodies validated for?

Currently available DCAF17 antibodies have been validated for several research applications:

Most commercially available DCAF17 antibodies are rabbit polyclonal antibodies generated against specific regions of the human DCAF17 protein . For example, Abcam's ab185324 is raised against amino acids 300-450, while other antibodies target regions such as amino acids 235-335 .

What reactivity do DCAF17 antibodies show across different species?

Species reactivity varies by antibody, with most validated primarily for human samples. Based on sequence homology and experimental validation:

• Human: All reviewed antibodies show reactivity

• Mouse: Several antibodies demonstrate reactivity

• Rat: Confirmed reactivity with some antibodies

Some manufacturers report potential cross-reactivity with additional species based on sequence homology. For example, Thermo Fisher Scientific's PA5-107109 antibody shows sequence homology with multiple species: Dog (100%), Guinea Pig (93%), Horse (100%), Mouse (100%), Pig (100%), Rabbit (100%), and Rat (100%) .

How should I validate DCAF17 antibody specificity for my experimental system?

Proper validation of DCAF17 antibody specificity requires a multi-faceted approach:

• Positive and negative controls:

  • Positive: Use tissues with known high expression (testis, brain) or validated cell lines (HEK-293, HL-60, RT-4, U-251 MG)

  • Negative: Include tissues with low expression (skin) or DCAF17 knockout/knockdown samples where available

• Molecular weight verification:

  • Confirm band appearance at expected molecular weight (theoretical: 59 kDa, though may run at ~68 kDa in some systems)

• Multiple antibody approach:

  • Use antibodies targeting different epitopes of DCAF17

  • Consistent results across different antibodies strengthen validation

• Peptide competition assay:

  • Pre-incubate antibody with immunizing peptide

  • Signal should be significantly reduced or eliminated

• Correlation with mRNA expression:

  • Compare protein detection patterns with DCAF17 mRNA expression data as reported in the literature

For developmental studies, be particularly mindful of DCAF17's age-dependent expression pattern in testis .

What are optimal protocols for Western blotting with DCAF17 antibodies?

For reliable Western blot detection of DCAF17:

• Sample preparation:

  • Standard lysis buffers with protease inhibitors are suitable

  • Include positive control samples (HEK-293, HL-60 cells, human liver tissue)

• SDS-PAGE conditions:

  • 10-12% gels provide optimal resolution around the expected 59-68 kDa range

  • Load 20-30 μg total protein per lane

• Antibody conditions:

  • Primary antibody dilutions: Follow manufacturer recommendations

    • ab185324: 1:500 dilution

    • 26033-1-AP: 1:500-1:2000

    • A306774: 1:500-1:2000

  • Overnight incubation at 4°C generally yields best results

• Expected results:

  • Predicted band size: 59 kDa (theoretical)

  • Observed molecular weight: May appear at 68 kDa

  • Validated positive samples include HEK-293 cells, HL-60 cells, RT-4 cells, U-251 MG cells, human liver tissue, and human tonsil tissue

Western blot results from Abcam (ab185324) demonstrate detection across multiple human samples:

What are the optimal protocols for immunohistochemistry with DCAF17 antibodies?

For immunohistochemical detection of DCAF17:

• Tissue preparation:

  • Formalin-fixed, paraffin-embedded (FFPE) tissues are suitable

  • Section thickness: 4-6 μm

• Antigen retrieval:

  • Critical for optimal staining

  • For 26033-1-AP: TE buffer pH 9.0 is recommended

  • Alternative: Citrate buffer pH 6.0

• Antibody conditions:

  • Dilution ranges:

    • ab185324: 1:1000

    • 26033-1-AP: 1:50-1:500

  • Incubation: Overnight at 4°C or 1-2 hours at room temperature

• Validated tissues:

  • Human cerebral cortex: Successfully labeled with ab185324 at 1:1000 dilution

  • Mouse liver: Positive with 26033-1-AP

  • Testis: Expected high expression based on mRNA data

• Optimization note:

  • Antibody concentration should be titrated for each testing system and tissue type to achieve optimal results

How can DCAF17 antibodies be used to study male fertility and spermatogenesis?

DCAF17 antibodies are valuable tools for investigating male fertility, as DCAF17 knockout studies have revealed its essential role in spermatogenesis:

• Immunohistochemical analysis of testicular tissue:

  • Track DCAF17 expression throughout postnatal testis development

  • Examine expression in specific cell types within seminiferous tubules

  • Compare normal versus infertility models

• Detection of specific spermatogenesis defects:

  • DCAF17 deletion causes specific defects detectable with microscopy and immunostaining:

    • Asymmetric acrosome capping

    • Impaired nuclear compaction

    • Abnormal round spermatid to elongated spermatid transition

    • Manchette formation defects

• Co-localization studies:

  • Combined immunofluorescence with markers for:

    • Acrosome (e.g., PNA-FITC staining)

    • Nuclear structures

    • Manchette proteins

• Quantitative analysis:

  • Monitor DCAF17 levels during testicular development

  • qRT-PCR data shows gradual increase in expression with age until 32 days postpartum

• Expression in infertility models:

  • Compare DCAF17 expression in normal versus infertile individuals

  • Potential diagnostic biomarker for specific forms of male infertility

The specificity of DCAF17 to male fertility (with no effect on female fertility) makes it particularly valuable for studying male-specific infertility mechanisms .

What is the relationship between DCAF17 and Woodhouse-Sakati syndrome?

DCAF17 antibodies can provide insights into Woodhouse-Sakati syndrome (WSS), a rare neuroendocrine disorder caused by DCAF17 mutations:

• Genetic basis:

  • WSS is caused by mutations in the DCAF17 gene

  • Multiple mutation types have been reported:

    • Nonsense mutations (e.g., c.1111delA, p.(Ile371Term))

    • Splice site mutations (e.g., c.321+1G>A)

    • Frameshift mutations (e.g., c.1238delA)

    • Start loss mutation (c.1G>A)

• Research applications:

  • Examine DCAF17 protein expression and localization in patient-derived cells

  • Investigate nucleolar structure and function in WSS patient samples

  • Study impact of specific DCAF17 mutations on protein stability and function

• Clinical manifestations:

  • DCAF17 antibodies can help investigate protein expression in tissues affected by WSS:

    • Hair follicles (alopecia)

    • Endocrine tissues (diabetes, hypogonadism)

    • Neural tissues (hearing loss, extrapyramidal symptoms)

    • Gonads (hypogonadism/gonadal agenesis)

• Molecular pathogenesis:

  • DCAF17 mutations lead to nucleolar dysfunction affecting:

    • Ribosome biogenesis

    • Cell cycle regulation

    • Cellular aging

    • RNA processing

    • mRNA transport

    • Apoptosis

Understanding the mechanistic relationship between DCAF17 mutations and WSS features may provide insights into therapeutic approaches for this rare disorder.

How can DCAF17 antibodies be utilized to study ubiquitination pathways?

As a component of the CUL4-DDB1 E3 ubiquitin ligase complex, DCAF17 antibodies can be employed to investigate ubiquitination pathways:

• Co-immunoprecipitation studies:

  • Immunoprecipitate DCAF17 and identify associated proteins by Western blotting or mass spectrometry

  • Detect interactions with:

    • Core complex components (CUL4, DDB1)

    • Potential substrate proteins

    • Other regulatory proteins

• Substrate identification:

  • Compare ubiquitinated protein profiles in normal versus DCAF17-depleted cells

  • Use sequential immunoprecipitation (DCAF17 followed by ubiquitin) to identify specific substrates

• Subcellular localization studies:

  • DCAF17 is reported to be a nucleolar protein

  • Immunofluorescence can track localization during:

    • Cell cycle progression

    • Stress responses

    • Spermatogenic differentiation

• Post-translational modification analysis:

  • Immunoprecipitate DCAF17 and analyze for:

    • Self-ubiquitination (potential regulatory mechanism)

    • Other modifications that may regulate function

• Structure-function studies:

  • Examine how deletions or mutations affect:

    • Complex formation with CUL4-DDB1

    • Substrate binding

    • Nucleolar localization

This research direction may uncover novel substrates of DCAF17-containing E3 ligase complexes and explain the tissue-specific effects of DCAF17 deficiency.

How can I troubleshoot non-specific binding when using DCAF17 antibodies?

When encountering non-specific binding with DCAF17 antibodies:

• For Western blotting issues:

  • Increase blocking time and concentration (5% to 10% blocking agent)

  • Add 0.1-0.3% Triton X-100 to reduce hydrophobic interactions

  • Increase washing duration and number of washes

  • Titrate primary antibody concentration

  • Consider that some bands may represent isoforms (alpha: 453 aa; beta: 520 aa)

  • Validate with knockout/knockdown controls when possible

• For immunohistochemistry/immunocytochemistry:

  • Optimize antigen retrieval conditions (compare citrate buffer pH 6.0 vs. TE buffer pH 9.0)

  • Titrate antibody concentration more extensively

  • Try longer blocking periods

  • Include additional blocking steps with species-specific serum

  • For mouse tissues on mouse antibodies, use specialized blocking reagents to reduce endogenous antibody binding

• For all applications:

  • Compare multiple DCAF17 antibodies targeting different epitopes

  • Include appropriate positive and negative controls

  • Consider batch-to-batch variation of antibodies

  • Validate with genetic approaches (siRNA, CRISPR) when possible

Careful optimization of each experimental step is essential for minimizing non-specific binding while maintaining sensitivity for DCAF17 detection.

What controls should be included when working with DCAF17 antibodies?

Proper experimental controls are critical for reliable DCAF17 research:

• Positive tissue/cell controls:

  • Testis (highest expression)

  • HEK-293 cells, HL-60 cells (validated for multiple antibodies)

  • RT-4 cells, U-251 MG cells

  • Human liver tissue, human tonsil tissue

• Negative controls:

  • Primary antibody omission

  • Isotype controls (rabbit IgG for rabbit polyclonal DCAF17 antibodies)

  • DCAF17 knockout/knockdown samples

  • Tissues with low expression (skin shows similar expression to brain)

• Peptide competition controls:

  • Pre-incubate antibody with immunizing peptide

  • Example immunogen sequences include:

    • Recombinant fragment within Human DCAF17 aa 300-450

    • Amino acids 235-335 of human DCAF17

    • EQETFKIVDYEDELDLLSVVAVTQIDAEGKAHLDFHCNEYGTLLKSIPLVESWDVTYSHEVYFDRDLVLHIEQKPNRVFSCYVYQMICD

• Technical controls:

  • For Western blot: Loading controls (β-actin, GAPDH, etc.)

  • For IHC/ICC: Background staining controls

  • For developmental studies: Age-matched controls (given age-dependent expression)

These comprehensive controls help ensure experimental rigor and reproducibility in DCAF17 research.

How might DCAF17 antibodies be utilized in identifying novel therapeutic targets?

DCAF17 antibodies could facilitate discovery of therapeutic targets through:

• Substrate identification:

  • Immunoprecipitation coupled with mass spectrometry to identify DCAF17-interacting proteins

  • Comparative proteomics between normal and DCAF17-deficient samples

  • Validation of potential substrates in relevant biological contexts

• Pathway analysis:

  • Investigation of DCAF17-regulated pathways in:

    • Spermatogenesis models

    • Neurological development

    • Endocrine function

    • Hair follicle biology (relevant to WSS)

• Biomarker development:

  • Evaluation of DCAF17 protein levels in:

    • Male infertility screening

    • Potential early detection of WSS features

    • Monitoring treatment responses

• Drug development opportunities:

  • Screening compounds that modulate DCAF17-substrate interactions

  • Developing strategies to stabilize mutant DCAF17 in WSS

  • Identifying compounds that bypass DCAF17 deficiency by targeting downstream effectors

These approaches could lead to novel therapeutic strategies for male infertility and Woodhouse-Sakati syndrome, conditions currently lacking effective treatments.

What unanswered questions remain regarding DCAF17 function?

Despite recent advances, several important questions about DCAF17 remain unanswered:

• Substrate specificity:

  • What are the specific substrates of DCAF17-containing E3 ligase complexes?

  • How does DCAF17 recognize its substrates?

  • Are there tissue-specific substrate preferences?

• Regulatory mechanisms:

  • How is DCAF17 expression and function regulated?

  • What post-translational modifications affect DCAF17 activity?

  • Does DCAF17 undergo self-regulation through auto-ubiquitination?

• Developmental roles:

  • Why does DCAF17 deletion specifically affect male fertility but not female fertility?

  • What explains the tissue-specific manifestations of WSS despite widespread expression?

  • Does DCAF17 have distinct functions during different developmental stages?

• Therapeutic implications:

  • Can restoration of DCAF17 function reverse pathology in disease models?

  • Are there compensatory mechanisms that could be therapeutically enhanced?

  • Could targeting DCAF17 substrates provide therapeutic benefits?

Addressing these questions will require sophisticated applications of DCAF17 antibodies in combination with genetic, biochemical, and cell biological approaches.