KCTD19 Antibody

What is KCTD19 and what is its primary biological function?

KCTD19 is an essential protein for meiosis, particularly in male gametogenesis. Research indicates that KCTD19 functions as a key transcriptional regulator required for the proper progression of meiotic prophase in spermatocytes. It associates with zinc finger protein 541 (ZFP541) and histone deacetylase 1 (HDAC1) to form a protein complex that regulates the transcriptional activity of meiotic genes . KCTD19 expression is primarily observed in the nuclei of spermatocytes during the early pachytene stage (seminiferous stage III-IV) and remains present through metaphase-anaphase transition and in round spermatids, before disappearing in elongating spermatids .

What types of KCTD19 antibodies are available for research applications?

Multiple types of KCTD19 antibodies have been validated for research purposes, including:

• Rabbit polyclonal antibodies (pAb), such as CAB17274

• Rat monoclonal antibodies (mAb), including:

  • mAb #19-3 (validated for immunoprecipitation)

  • mAb #22-15 (validated for immunostaining)

These antibodies have been confirmed to specifically recognize KCTD19 through validation experiments including western blotting with KCTD19 knockout models .

What are the optimal applications for KCTD19 antibodies in reproductive biology research?

KCTD19 antibodies have been successfully employed in multiple experimental applications:

The choice of antibody should be guided by the specific experimental application, with immunostaining studies benefiting from the higher specificity of the monoclonal antibody #22-15, while immunoprecipitation experiments can utilize either rabbit pAb or rat mAb #19-3 .

What is the recommended protocol for immunoprecipitation using KCTD19 antibodies?

For optimal immunoprecipitation of KCTD19 and its associated proteins, the following methodology has been validated:

• Prepare testis lysate using non-ionic detergent (NP40) buffer containing protease inhibitors

• Incubate the lysate with KCTD19 antibodies (either rabbit pAb or rat mAb #19-3) overnight at 4°C

• Add protein G-conjugate beads and incubate for 4 hours at 4°C on an orbital shaker

• Wash the beads thoroughly to remove non-specific binding

• Elute the protein complexes and analyze by SDS-PAGE followed by silver staining or western blotting

This protocol has successfully identified KCTD19's interactions with HDAC1 and ZFP541, confirming the formation of a protein complex involved in meiotic regulation .

How should researchers optimize western blotting protocols when using KCTD19 antibodies?

For effective western blotting detection of KCTD19:

• Prepare protein samples from testicular tissue or transfected cells using standard extraction methods

• Separate proteins using SDS-PAGE and transfer to PVDF membranes

• Block membranes with appropriate blocking buffer

• Incubate with anti-KCTD19 antibody at 1:1000 dilution overnight at 4°C

• Use goat anti-rabbit IgG HRP-conjugated secondary antibodies (1:2000) for 1.5 hours at 25°C

• Visualize using enhanced chemiluminescence

• Quantify relative KCTD19 levels using ImageJ2 software, normalizing to GAPDH as an internal control

This method has been successfully employed to assess KCTD19 variant expression and stability in comparative studies .

What controls should be included when validating KCTD19 antibody specificity?

Proper validation of KCTD19 antibody specificity requires the following controls:

• Genetic knockout control: Testicular tissues from KCTD19 knockout mice provide the gold standard negative control, confirming complete loss of signal in western blotting and immunostaining applications

• Multiple antibody validation: Using different antibodies (polyclonal and monoclonal) targeting different epitopes of KCTD19 to confirm consistent detection patterns

• Peptide competition assay: Pre-incubation of the antibody with excess KCTD19 peptide should abolish specific signal

• Positive controls: Using tissues with known KCTD19 expression (testis) versus tissues lacking KCTD19 expression

Research has validated antibody specificity using KCTD19 knockout mice, demonstrating complete loss of KCTD19 signal in knockout testis tissues compared to heterozygous or wild-type controls .

How can KCTD19 antibodies be employed for studying protein-protein interactions in meiotic research?

For investigating KCTD19's interactions with partner proteins:

• Co-immunoprecipitation coupled with mass spectrometry:

  • Perform immunoprecipitation with KCTD19 antibodies from testicular lysates

  • Subject eluted proteins to mass spectrometry analysis

  • This approach has successfully identified ZFP541 and HDAC1 as KCTD19-interacting proteins

• Reciprocal immunoprecipitation:

  • Confirm protein interactions by performing reverse immunoprecipitation with antibodies against potential interacting partners (e.g., anti-HDAC1)

  • Western blot for KCTD19 in the immunoprecipitated sample

• Co-localization studies:

  • Co-transfect cells with tagged constructs (e.g., FLAG-tagged KCTD19 and MYC-tagged ZFP541)

  • Perform immunofluorescence to assess cellular co-localization

  • This approach revealed that wild-type KCTD19 and ZFP541 show strong nuclear co-localization (38.7% ± 8.1%), while mutant KCTD19 variants exhibit significantly reduced nuclear co-localization

What methodological approaches can detect changes in KCTD19 protein stability and degradation?

To investigate KCTD19 protein stability and degradation mechanisms:

• Ubiquitination assays:

  • Co-express KCTD19 (wild-type or mutant) with tagged ubiquitin in cell culture

  • Immunoprecipitate KCTD19 and probe for ubiquitin modifications

  • This approach revealed that KCTD19 mutants (p.E210K, p.P298L, p.G770D) show increased ubiquitin conjugation, indicating accelerated protein degradation

• Protein half-life analysis:

  • Treat cells expressing KCTD19 with cycloheximide to inhibit new protein synthesis

  • Collect samples at different time points and analyze KCTD19 levels by western blotting

  • Calculate protein half-life by quantifying the rate of KCTD19 degradation

• Proteasome inhibition:

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

  • Assess whether KCTD19 protein levels are rescued, confirming proteasomal degradation

How should researchers design experiments to study the functional impact of KCTD19 mutations?

For comprehensive assessment of KCTD19 mutations:

• Expression constructs:

  • Generate wild-type and mutant KCTD19 expression plasmids (e.g., p.E210K, p.P298L, p.G770D)

  • Transfect into appropriate cell lines (e.g., HEK293T cells)

• Subcellular localization:

  • Perform immunofluorescence to determine whether mutations affect KCTD19 localization

  • Research has shown that while both wild-type and mutant KCTD19 localize primarily to the cytoplasm in HEK293T cells, mutations can affect nuclear translocation when co-expressed with ZFP541

• Protein-protein interaction:

  • Conduct co-immunoprecipitation experiments with wild-type versus mutant KCTD19

  • Assess whether mutations disrupt interactions with key partners like ZFP541

  • Findings indicate that mutations p.E210K and p.P298L weaken the interaction with ZFP541

• Functional rescue experiments:

  • Test whether transgenic expression of wild-type KCTD19 can rescue phenotypes in knockout models

  • This approach confirmed that testis-specific expression of epitope-tagged KCTD19 under the Clgn promoter can restore fertility in Kctd19 knockout mice

How can researchers address non-specific binding issues when using KCTD19 antibodies?

Non-specific binding can be minimized through several approaches:

• Optimize blocking conditions:

  • Test different blocking agents (BSA, non-fat milk, commercial blockers)

  • Extend blocking time to reduce background signal

• Antibody dilution optimization:

  • Perform a titration series to determine optimal antibody concentration

  • The recommended 1:1000 dilution for western blotting provides a balance between specific signal and background

• Cross-adsorption:

  • Pre-adsorb antibodies with tissues from KCTD19 knockout mice to remove non-specific antibodies

  • This is particularly useful for immunohistochemistry applications

• Use monoclonal antibodies:

  • For applications requiring higher specificity, use characterized monoclonal antibodies like rat mAb #22-15 for immunostaining

What are potential explanations for discrepancies between KCTD19 protein levels detected in vitro versus in vivo models?

Several factors may explain discrepancies between in vitro and in vivo KCTD19 detection:

• Cell-type specific post-translational modifications:

  • KCTD19 may undergo different post-translational modifications in testicular cells versus heterologous expression systems

  • This could affect antibody epitope recognition or protein stability

• Protein-protein interactions:

  • In testicular cells, KCTD19 naturally interacts with partners like ZFP541 and HDAC1

  • These interactions may stabilize the protein or affect its localization, as demonstrated by the increased nuclear localization of KCTD19 when co-expressed with ZFP541

• Differences in degradation pathways:

  • The ubiquitin-proteasome system may function differently across cell types

  • Research has shown that KCTD19 mutants undergo accelerated ubiquitin-mediated degradation in HEK293T cells

• Expression level differences:

  • Overexpression in heterologous systems may overwhelm normal degradation pathways

  • This could lead to artificial accumulation or mislocalization of the protein

How can KCTD19 antibodies be utilized to study male infertility phenotypes?

KCTD19 antibodies offer valuable tools for investigating male infertility through:

• Diagnostic immunohistochemistry:

  • Analyze KCTD19 expression patterns in testicular biopsies from infertile men

  • Compare with normal controls to identify potential defects in expression or localization

• Phenotype-genotype correlation studies:

  • Perform immunostaining on testicular samples from patients with identified KCTD19 variants

  • Assess whether mutations affect protein expression, localization, or stability

  • Research indicates that patients with homozygous KCTD19 missense variants exhibit oligoasthenoteratozoospermia rather than complete azoospermia observed in knockout mice

• Functional domain mapping:

  • Use domain-specific antibodies to investigate how different KCTD19 domains contribute to protein function

  • This approach could help explain why mutations in different domains (e.g., BTB_POZ domain versus C-terminal region) result in similar functional consequences

What experimental approaches can reconcile the phenotypic differences between human KCTD19 mutations and mouse knockout models?

To address the phenotypic discrepancy between human mutations (oligozoospermia) and mouse knockouts (azoospermia):

• Generation of knock-in mouse models:

  • Create mice harboring the specific human mutations (p.E210K, p.P298L, p.G770D)

  • Compare spermatogenesis phenotypes with complete knockout models

  • Assess whether partial protein function is retained in missense mutations

• Quantitative proteomic analysis:

  • Compare KCTD19 protein levels in mutant versus wild-type cells

  • Determine whether mutations cause complete or partial loss of protein

  • Research suggests human mutations may result in reduced but not abolished protein activity

• Developmental timing analysis:

  • Track spermatogenesis progression in detail across different developmental stages

  • Assess whether species-specific factors influence the timing or severity of meiotic defects

  • This could explain why some human spermatocytes may complete meiosis despite KCTD19 mutations

• Environmental and genetic modifier studies:

  • Investigate whether additional genetic or environmental factors modify the phenotypic expression of KCTD19 deficiency in humans versus mice