FBXL19 Antibody

What is FBXL19 and why is it significant for research?

FBXL19 is a nuclear protein that functions as a CpG island-binding protein in mouse embryonic stem (ES) cells through its ZF-CxxC domain. It plays a critical role in recruiting the CDK-Mediator complex to CpG island-associated promoters of non-transcribed developmental genes, priming these genes for activation during cell lineage commitment. Recognition of CpG islands by FBXL19 has been shown to be essential for mouse development. Additionally, FBXL19 functions as a component of an SCF (Skp, Cullin, F-box) E3 ligase complex with anti-inflammatory effects, controlling actin cytoskeleton dynamics by targeting cell membrane receptors and small GTPases for ubiquitination and degradation .

What are the key considerations when selecting an FBXL19 antibody?

When selecting an FBXL19 antibody, researchers should consider: (1) The specific application (Western blot, ChIP, immunofluorescence, or immunoprecipitation), as antibodies perform differently across techniques; (2) The species reactivity needed, as the search results indicate work with both mouse and human FBXL19; (3) The target epitope, as FBXL19 contains distinct domains including the ZF-CxxC domain, F-box domain, and leucine-rich repeats; (4) Monoclonal versus polyclonal options—monoclonals offer specificity while polyclonals may provide stronger signals; and (5) Validation data availability, especially given that some commercially available FBXL19 antibodies have shown unreliable performance in Western blot analysis as noted in the research .

How is FBXL19 protein regulated at the post-translational level?

FBXL19 is regulated through a balance between ubiquitination and acetylation. It has a relatively short half-life of approximately 3 hours. FBXL19 can be polyubiquitinated, and treatment with the proteasome inhibitor MG-132 prolongs its half-life, indicating that FBXL19 degradation occurs via the ubiquitin-proteasome system rather than lysosomal degradation. Importantly, FBXL19 can also be acetylated, and this acetylation reduces its ubiquitination levels, thereby increasing protein stability. The histone acetyltransferase CBP has been identified as an enzyme that catalyzes FBXL19 acetylation. Inhibition or downregulation of CBP reduces FBXL19 stability, whereas CBP overexpression increases it. This regulatory mechanism impacts the function of SCF FBXL19 E3 ligase by controlling the availability of its F-box protein subunit .

What are the validated applications for FBXL19 antibodies in research?

Based on the search results, validated applications for FBXL19 antibodies include: (1) Chromatin immunoprecipitation followed by sequencing (ChIP-seq) to identify genome-wide binding sites at CpG islands; (2) Immunofluorescence staining to determine subcellular localization; (3) Immunoprecipitation to study protein-protein interactions, particularly with components of the CDK-Mediator complex; (4) Biochemical fractionation experiments to determine subcellular distribution; and (5) Western blot analysis, though with some limitations as noted in the research. Some researchers have overcome detection issues by tagging endogenous FBXL19 with epitope tags (3xT7-2xStrepII) using CRISPR/Cas9 knock-in approaches .

How can FBXL19 antibodies be effectively used in ChIP-seq experiments?

For effective ChIP-seq with FBXL19 antibodies, researchers should: (1) Consider using epitope-tagged FBXL19 (as described in the search results) if antibody performance is suboptimal; (2) Include appropriate controls, such as input DNA and IgG ChIP; (3) Compare FBXL19 binding with known CpG island markers such as KDM2A and KDM2B; (4) Correlate binding profiles with non-methylated CpG density; and (5) Validate findings using mutant versions of FBXL19 (e.g., K49A ZF-CxxC domain mutant or ΔCXXC deletion mutant) to confirm binding specificity. The search results describe successful ChIP-seq experiments that revealed FBXL19 enrichment at both computationally predicted CpG islands and regions containing BioCAP signal (an experimental measure of non-methylated DNA) .

What methods are recommended for studying FBXL19-mediated protein ubiquitination?

For studying FBXL19-mediated ubiquitination, researchers should consider: (1) Implementing a modified immunoprecipitation protocol under denaturing conditions as described in the search results, where cells are lysed in 2% SDS buffer containing ubiquitin aldehyde and N-ethylmaleimide (to inhibit deubiquitinases); (2) Sonicating and boiling samples to denature proteins before dilution with PBS; (3) Following with standard immunoprecipitation procedures; (4) Including proteasome inhibitors (e.g., MG-132) in experimental designs to accumulate ubiquitinated substrates; (5) Comparing ubiquitination levels in the presence or absence of FBXL19 overexpression or knockdown; and (6) Analyzing specific substrates such as Cdc42, which has been identified as an FBXL19 target for ubiquitination and degradation .

What are the optimal conditions for immunoprecipitation of FBXL19?

For optimal immunoprecipitation of FBXL19, researchers should: (1) Use appropriate cell lysis buffers that preserve protein-protein interactions; (2) Include protease inhibitors to prevent degradation; (3) Consider that FBXL19 interactions with some proteins, such as components of the CDK-Mediator complex, appear to be sub-stoichiometric and relatively weak, as noted in the search results; (4) Use sufficient starting material (the search results indicate using 1 mg of cell lysate); (5) Allow adequate incubation time (overnight at 4°C with antibody followed by 2 hours with protein A/G agarose); and (6) Perform thorough washing steps (3 times with 1% Triton X-100 in ice-cold PBS). If direct immunoprecipitation proves challenging due to antibody limitations, consider using epitope-tagged versions of FBXL19 or creating CRISPR/Cas9 knock-in cell lines with endogenously tagged FBXL19 .

What controls should be included in FBXL19 antibody experiments?

Critical controls for FBXL19 antibody experiments include: (1) Isotype-matched IgG controls for immunoprecipitation and ChIP experiments; (2) FBXL19 knockdown or knockout cells as negative controls; (3) Cells overexpressing FBXL19 as positive controls; (4) For ChIP-seq studies, include mutant versions of FBXL19 (K49A and ΔCXXC) to validate binding specificity; (5) For studies of FBXL19 stability, include cycloheximide chase experiments with and without proteasome inhibitors; (6) For studies of FBXL19 acetylation, include deacetylase inhibitors (e.g., TSA) and acetyltransferase inhibitors (e.g., C646, anacardic acid); and (7) For functional studies, include both wild-type and mutant versions of FBXL19 to distinguish specific effects from non-specific ones .

How should samples be prepared for optimal FBXL19 western blot detection?

For optimal FBXL19 western blot detection: (1) Note that some commercially available FBXL19 antibodies have shown unreliable performance in Western blot analysis, as mentioned in the search results; (2) Consider generating cell lines expressing epitope-tagged FBXL19 (FS2-FBXL19) or creating CRISPR/Cas9 knock-in cell lines with endogenously tagged FBXL19 (3xT7-2xStrepII tag); (3) Use appropriate lysis buffers and include protease inhibitors to prevent degradation; (4) Include phosphatase inhibitors if studying phosphorylation states; (5) Given FBXL19's relatively short half-life (~3 hours), consider proteasome inhibitor treatment to stabilize the protein before lysis; (6) Use freshly prepared samples when possible, as FBXL19 may be unstable during storage; and (7) For detection of specific post-translational modifications, consider using antibodies against those modifications (e.g., anti-acetylated lysine) followed by immunoprecipitation of FBXL19 .

How can researchers investigate the role of FBXL19 in CpG island binding and gene regulation?

To investigate FBXL19's role in CpG island binding and gene regulation, researchers can: (1) Perform ChIP-seq with FBXL19 antibodies or epitope-tagged FBXL19 to identify genome-wide binding sites; (2) Generate ZF-CxxC domain mutants (K49A) or deletion mutants (ΔCXXC) to assess the dependency of binding on this domain; (3) Correlate FBXL19 binding with non-methylated CpG density and other CpG island-binding proteins (e.g., KDM2A, KDM2B); (4) Integrate FBXL19 binding data with transcriptome data to identify regulated genes; (5) Perform ChIP-seq for components of the CDK-Mediator complex to assess co-localization with FBXL19; (6) Utilize FBXL19 knockout or knockdown approaches followed by RNA-seq to identify genes regulated by FBXL19; and (7) Study the dynamics of FBXL19 binding during cellular differentiation to understand its role in developmental gene regulation .

What approaches can be used to study the interaction between FBXL19 and the CDK-Mediator complex?

To study FBXL19-CDK-Mediator interactions, researchers should: (1) Perform co-immunoprecipitation experiments using antibodies against FBXL19 and CDK-Mediator components (e.g., MED12, CDK8) as described in the search results; (2) Use mass spectrometry analysis of affinity-purified FBXL19 complexes to identify interacting partners; (3) Recognize that the interaction appears to be sub-stoichiometric and relatively weak, which may require optimized conditions for detection; (4) Consider using protein crosslinking approaches to stabilize transient interactions; (5) Perform ChIP-seq for both FBXL19 and CDK-Mediator components to assess co-occupancy at genomic sites; (6) Generate domain mutants of FBXL19 to map the interaction interface with CDK-Mediator; and (7) Employ functional assays, such as reporter gene assays, to assess the impact of FBXL19 on CDK-Mediator-dependent transcription .

How can researchers investigate the role of FBXL19 acetylation in protein stability and function?

To investigate FBXL19 acetylation: (1) Perform cycloheximide chase experiments in the presence or absence of deacetylase inhibitors (e.g., TSA) to assess protein stability; (2) Use acetylation-mimic mutants (lysine to glutamine substitution) or acetylation-deficient mutants (lysine to arginine substitution) to study the impact on protein stability and function; (3) Investigate the role of CBP in FBXL19 acetylation through overexpression, knockdown, or inhibition (e.g., with C646); (4) Perform mass spectrometry analysis to identify specific acetylation sites; (5) Assess the impact of acetylation on FBXL19 ubiquitination using the in vivo ubiquitin assay described in the search results; (6) Investigate whether acetylation affects FBXL19's interaction with other SCF components or its substrate recognition; and (7) Study the functional consequences of altered FBXL19 stability on its targets, such as Cdc42 ubiquitination and degradation .

Why might FBXL19 antibodies show inconsistent results in western blotting?

FBXL19 antibodies may show inconsistent Western blot results due to: (1) The relatively short half-life of FBXL19 (~3 hours), leading to low endogenous levels; (2) Potential post-translational modifications affecting epitope recognition; (3) The search results specifically note that "the FBXL19 antibody failed to work reliably for Western blot analysis," suggesting inherent challenges with available antibodies; (4) Variation in antibody quality between lots or manufacturers; (5) Suboptimal sample preparation conditions for preserving FBXL19; (6) Differences in FBXL19 expression levels between cell types; and (7) Technical variations in transfer efficiency or detection methods. Researchers have overcome these limitations by generating epitope-tagged versions of FBXL19 or creating CRISPR/Cas9 knock-in cell lines with endogenously tagged FBXL19 .

What strategies can improve detection of endogenous FBXL19 in cells with low expression?

To improve detection of endogenous FBXL19: (1) Treat cells with proteasome inhibitors (e.g., MG-132) to stabilize FBXL19 protein; (2) Use deacetylase inhibitors (e.g., TSA) to enhance acetylation and reduce ubiquitination; (3) Consider generating CRISPR/Cas9 knock-in cell lines with endogenously tagged FBXL19 as described in the search results; (4) Implement more sensitive detection methods, such as enhanced chemiluminescence (ECL) or fluorescence-based detection; (5) Enrich FBXL19 through immunoprecipitation before Western blot analysis; (6) Optimize lysis conditions to ensure complete extraction of nuclear proteins, as FBXL19 is predominantly nuclear; and (7) Concentrate samples through techniques such as TCA precipitation if necessary .

How can researchers troubleshoot failed FBXL19 chromatin immunoprecipitation experiments?

For troubleshooting failed FBXL19 ChIP experiments: (1) Verify antibody specificity and ChIP compatibility through preliminary tests; (2) Consider using epitope-tagged FBXL19 if antibody performance is suboptimal; (3) Optimize crosslinking conditions, as FBXL19 is a DNA-binding protein with potentially transient interactions; (4) Ensure complete chromatin shearing to appropriate fragment sizes; (5) Increase the amount of starting material, as FBXL19 may be present at low levels; (6) Optimize wash stringency to balance between signal retention and background reduction; (7) Include positive controls such as ChIP for known CpG island-binding proteins (e.g., KDM2A, KDM2B); and (8) Validate ChIP-seq findings using alternative methods, such as comparing wild-type FBXL19 with ZF-CxxC domain mutants (K49A) or deletion mutants (ΔCXXC) .

What is the comparative stability of wild-type versus mutant FBXL19 proteins?

This table summarizes key findings regarding FBXL19 protein stability based on the search results, highlighting how different variants respond to proteasome inhibition (MG-132) and deacetylase inhibition (TSA) .

What are the key domain-specific functions of FBXL19?

This table organizes domain-specific functions of FBXL19 based on experimental evidence from the search results, providing a comprehensive view of structure-function relationships .

What methods have been validated for studying FBXL19 post-translational modifications?

This table summarizes validated methods for studying post-translational modifications of FBXL19 based on the search results, providing researchers with experimental approaches and appropriate controls .