KCTD7 Antibody, FITC conjugated

What is KCTD7 and why is it important in neuroscience research?

KCTD7 (potassium channel tetramerization domain-containing protein 7) is a member of the KCTD protein family that plays essential roles in neuronal function and development. Despite its name suggesting involvement with potassium channels, KCTD family members lack predicted channel domains and instead function primarily in other cellular processes. KCTD7 is particularly significant because mutations in this gene are associated with rare autosomal recessive disorders including progressive myoclonic epilepsy (EPM3) and neuronal ceroid lipofuscinosis type 14 (CLN14) . The protein has been shown to be essential for proper neuronal function and development, making it a potential target for understanding neurological disorders and brain function .

At the molecular level, KCTD7 functions as an adaptor of the CUL3-RING E3 ubiquitin ligase (CRL3) complex, participating in protein degradation pathways critical for neuronal homeostasis . Research has demonstrated that KCTD7 is involved in the control of excitability of cortical neurons , underscoring its importance in neuroscience investigations focusing on neural circuit function and neurodegeneration.

What are the recommended applications for FITC-conjugated KCTD7 antibodies?

FITC-conjugated KCTD7 antibodies are primarily recommended for immunofluorescence (IF) applications, with optimal dilution ranges of 1:50-1:200 . These antibodies are particularly valuable for visualizing the subcellular localization of KCTD7 in fixed cells and tissue sections. In addition to immunofluorescence, non-conjugated versions of KCTD7 antibodies have been validated for ELISA applications at dilutions of 1:2000-1:10000 .

When utilizing FITC-conjugated KCTD7 antibodies for immunofluorescence, researchers should follow standard protocols for cell fixation (4% formaldehyde), permeabilization (0.2% Triton X-100), and blocking (10% normal goat serum) . The direct fluorescent conjugation eliminates the need for secondary antibody incubation steps, streamlining experimental workflows and reducing background issues that can occur with indirect immunofluorescence methods.

How should I validate the specificity of KCTD7 antibody staining?

Validating the specificity of KCTD7 antibody staining requires multiple complementary approaches:

First, perform immunoblotting comparison between samples with known KCTD7 expression (transfected cells) and negative controls (untransfected cells). Specific immunoreactive bands corresponding to KCTD7 should be present only in positive samples . Multiple bands may be observed due to alternative splice variants and potential proteolytic cleavage products .

Second, conduct peptide competition assays in which the antibody is pre-incubated with its peptide antigen (recombinant KCTD7 protein, amino acids 5-152) before application to samples. This should abolish specific staining if the antibody is truly specific .

Third, implement genetic controls by comparing staining patterns in wild-type cells versus KCTD7-deficient cells generated through CRISPR-Cas9 or siRNA approaches. Proper validation would show signal reduction or pattern changes in knockout/knockdown samples.

Finally, compare staining patterns across multiple KCTD7 antibodies recognizing different epitopes, which should yield consistent localization patterns if each antibody is specific .

What subcellular localization pattern should I expect when using KCTD7 antibodies?

When using KCTD7 antibodies for immunofluorescence, you should expect to observe a characteristic subcellular localization pattern that includes both cytoplasmic and plasma membrane signals. Studies with GFP-tagged wild-type KCTD7 have demonstrated a broad, somewhat punctate cytoplasmic distribution with distinct localization at the plasma membrane .

It's important to note that KCTD7 localization appears to be affected by mutations. For example, the patient-derived p.Arg184Cys mutation results in more diffuse cytoplasmic localization, markedly diminished plasma membrane signal, and the appearance of prominent cytoplasmic aggregates . Similarly, other disease-associated mutations in the BTB domain (T64A, L108M, D115Y) have been shown to dramatically alter the subcellular distribution pattern of KCTD7 .

When visualizing KCTD7 using immunofluorescence, counterstaining with DAPI to visualize nuclei is recommended to provide context for cytoplasmic and membrane staining . Confocal microscopy may be necessary to clearly distinguish membrane localization from cytoplasmic signals.

How can I use KCTD7 antibodies to investigate its role in protein degradation pathways?

KCTD7 functions as an adaptor of the CUL3-RING E3 ubiquitin ligase (CRL3) complex, which is involved in protein degradation through the ubiquitin-proteasome system . To investigate this role using KCTD7 antibodies, researchers can implement several methodological approaches:

First, perform co-immunoprecipitation (co-IP) experiments using KCTD7 antibodies to pull down protein complexes, followed by immunoblotting for CUL3, RBX1, and other components of the E3 ligase machinery. This can verify the interaction between KCTD7 and these proteins in your experimental system .

Second, conduct comparative ubiquitination assays in wild-type and KCTD7-deficient cells using KCTD7 antibodies alongside ubiquitin antibodies to assess changes in substrate ubiquitination. Research has shown that the CRL3-KCTD7 complex degrades CLN5, making this protein a prime candidate for such studies .

Third, use FITC-conjugated KCTD7 antibodies in combination with antibodies against CLN5 and other potential substrates for co-localization studies, which can provide spatial information about where these degradation events occur within the cell.

Finally, implement a proximity ligation assay (PLA) using KCTD7 antibodies and antibodies against ubiquitination machinery components to visualize the sites of active complex formation with single-molecule resolution.

What methods can I use to study the relationship between KCTD7 and lysosomal function?

Mutations in KCTD7 are associated with neuronal ceroid lipofuscinosis (NCL), a lysosomal storage disorder characterized by intracellular accumulation of ceroid in neurons . To investigate the relationship between KCTD7 and lysosomal function using KCTD7 antibodies, consider these methodological approaches:

First, perform co-localization studies using FITC-conjugated KCTD7 antibodies alongside markers for lysosomes (LAMP1, LAMP2) to determine whether KCTD7 associates with lysosomal compartments under normal and stress conditions.

Second, conduct comparative lysosomal enzyme trafficking assays in wild-type versus KCTD7-deficient cells. Research has shown that accumulated CLN5 (resulting from KCTD7 deficiency) disrupts the interaction between CLN6/8 and lysosomal enzymes at the endoplasmic reticulum, impairing ER-to-Golgi trafficking of lysosomal enzymes .

Third, use FITC-conjugated KCTD7 antibodies in pulse-chase experiments to track dynamic changes in KCTD7 localization in response to lysosomal stress or autophagy induction.

Fourth, implement electron microscopy with immunogold labeling using KCTD7 antibodies to visualize KCTD7 localization at the ultrastructural level, particularly in relation to lysosomal and autophagosomal structures that are abnormal in KCTD7-deficient cells .

How can I optimize double immunofluorescence protocols using FITC-conjugated KCTD7 antibodies?

When designing double immunofluorescence experiments with FITC-conjugated KCTD7 antibodies, several optimization steps are crucial:

First, select complementary fluorophores for the second antibody that have minimal spectral overlap with FITC (excitation 495nm, emission 520nm). Good options include far-red fluorophores like Cy5 or Alexa Fluor 647.

Second, determine the optimal fixation and permeabilization conditions. Standard protocols using 4% formaldehyde for fixation and 0.2% Triton X-100 for permeabilization have proven effective for KCTD7 antibodies , but these may need adjustment based on the requirements of the second antibody.

Third, optimize antibody concentrations individually before combining them. For FITC-conjugated KCTD7 antibodies, start with the recommended dilution range (1:50-1:200) and adjust based on signal-to-noise ratio.

Fourth, implement a sequential staining protocol if the second antibody is from the same host species as the KCTD7 antibody (typically rabbit). This involves complete blocking between applications of the first and second primary antibodies.

Finally, include appropriate controls: single-antibody stains to assess bleed-through, secondary-only controls to evaluate non-specific binding, and whenever possible, KCTD7-deficient samples as negative controls for staining specificity.

What approach should I take to study KCTD7 in patient-derived cells using antibodies?

Studying KCTD7 in patient-derived cells requires careful methodological considerations:

First, establish baseline expression and localization patterns of KCTD7 in control cells using FITC-conjugated KCTD7 antibodies. Wild-type KCTD7 typically shows broad cytoplasmic distribution with plasma membrane localization .

Second, compare these patterns with patient-derived cells carrying KCTD7 mutations. Different mutations cause distinct localization patterns - for example, the T64A mutation results in predominantly nuclear localization in a ball-and-stick pattern, while the L108M mutation increases mini-circles at filament termini, and D115Y causes massive filament-like structures .

Third, conduct functional assays to assess the impact of patient mutations on KCTD7's role in protein degradation. Co-immunoprecipitation experiments can determine whether mutations disrupt the interaction between KCTD7 and its partners (CUL3, CLN5) .

Fourth, implement live-cell imaging in patient cells transfected with fluorescently-tagged wild-type KCTD7 to assess whether the mutant cellular environment affects the behavior of the wild-type protein.

Finally, use KCTD7 antibodies to assess changes in downstream pathways affected by KCTD7 mutations, particularly lysosomal enzyme trafficking and autophagy, which show defects in KCTD7-deficient cells .

How can I use KCTD7 antibodies to investigate autophagic defects in neurological disorders?

KCTD7 deficiency leads to several autophagic defects, including accumulation of late autophagosome-like structures, increases in p62, ubiquitin, and LC3B puncta, and defective autophagic flux . To investigate these phenomena using KCTD7 antibodies:

First, implement triple immunofluorescence using FITC-conjugated KCTD7 antibodies alongside markers for autophagosomes (LC3B) and autolysosomes/lysosomes (LAMP1/2) to assess co-localization patterns and potential disruptions in the autophagy pathway.

Second, conduct comparative immunoblotting for autophagy markers (p62, LC3B-II) in control versus KCTD7-deficient or mutant cells. Reintroduction of wild-type KCTD7, but not ΔBTB mutant KCTD7, can reverse the accumulation of these markers, providing a functional rescue experiment to validate antibody-based findings .

Third, use KCTD7 antibodies in proximity ligation assays with autophagy-related proteins to identify direct molecular interactions that may be disrupted in disease states.

Fourth, implement live-cell imaging approaches with FITC-conjugated KCTD7 antibodies delivered via cell-penetrating peptides to track dynamic changes in KCTD7 localization during autophagy induction and flux in real-time.

Finally, correlate antibody-based findings with functional autophagy assays such as the mCherry-GFP-LC3B reporter assay, which has demonstrated that KCTD7 deficiency increases both autophagosome and autolysosome formation .

What methodological considerations are important when using KCTD7 antibodies to study its interaction with CLN5?

Research has identified CLN5 as a high-confidence interactor of KCTD7, with the CRL3-KCTD7 complex regulating CLN5 degradation . When studying this interaction using antibody-based methods:

First, perform reciprocal co-immunoprecipitation experiments using both KCTD7 and CLN5 antibodies to confirm direct interaction. This technique has successfully demonstrated the binding of KCTD7 with CLN5 in previous studies .

Second, design domain mapping experiments using truncated versions of KCTD7 and CLN5 to identify the specific regions mediating their interaction. The BTB/POZ domain (amino acids 5-152) used as an immunogen for many KCTD7 antibodies may be particularly relevant, as patient mutations in this region disrupt protein interactions.

Third, implement antibody-based competition assays to determine whether patient-derived KCTD7 mutations disrupt the KCTD7-CLN5 interaction. Research has shown that such mutations can disrupt interactions between KCTD7-CUL3 or KCTD7-CLN5 .

Fourth, use FITC-conjugated KCTD7 antibodies alongside CLN5 antibodies for super-resolution microscopy to visualize the spatial relationship between these proteins at nanometer resolution.

Finally, conduct pulse-chase experiments with cycloheximide to assess CLN5 stability in the presence of wild-type versus mutant KCTD7, using antibodies against both proteins to track their levels over time.

How can I detect and characterize different functional domains of KCTD7 using antibody-based methods?

KCTD7 contains several functional domains, including the N-terminal BTB/POZ domain and regions involved in protein-protein interactions. To characterize these domains using antibodies:

First, select antibodies targeting different epitopes within KCTD7. Many commercial antibodies recognize the BTB/POZ domain (amino acids 5-152) , but additional antibodies targeting other regions would allow more comprehensive domain analysis.

Second, implement domain-specific co-immunoprecipitation experiments to identify region-specific binding partners. The BTB/POZ domain is known to mediate interactions with CUL3 , but evidence points to the role of critical residues outside this domain as well .

Third, use FITC-conjugated KCTD7 antibodies to visualize domain-specific localization patterns. Research has shown that expression of the N-terminal amino acids 1-149 of wild-type KCTD7 (containing the BTB domain) forms unusual flowing filament-like structures in the cytoplasm and smaller structures in the nucleus .

Fourth, conduct antibody-based mutagenesis studies targeting specific domains. For example, compare antibody detection of wild-type KCTD7 versus the ΔBTB mutant to assess how this deletion affects epitope accessibility and protein stability.

Finally, implement limited proteolysis experiments followed by immunoblotting with domain-specific antibodies to assess conformational changes induced by mutations or binding partner interactions.

What strategies can I use to investigate the role of KCTD7 in regulating lysosomal enzyme trafficking?

KCTD7 deficiency leads to impaired ER-to-Golgi trafficking of lysosomal enzymes, mediated through accumulated CLN5 disrupting interactions between CLN6/8 and lysosomal enzymes . To investigate this phenomenon using antibody-based methods:

First, implement co-immunoprecipitation experiments using KCTD7 antibodies to pull down complexes containing CLN5, CLN6, and CLN8, and assess how KCTD7 mutations affect these interactions.

Second, conduct subcellular fractionation followed by immunoblotting with KCTD7 antibodies to track the distribution of KCTD7 and lysosomal enzymes across different cellular compartments (ER, Golgi, lysosomes) in control versus disease models.

Third, use FITC-conjugated KCTD7 antibodies in combination with antibodies against lysosomal enzymes for high-resolution co-localization studies to visualize trafficking defects at different stages of the secretory pathway.

Fourth, implement live-cell imaging with fluorescently-tagged lysosomal enzymes in cells with normal versus deficient KCTD7 levels (verified by immunoblotting) to track trafficking kinetics in real-time.

Finally, correlate antibody-based findings with functional assays measuring lysosomal enzyme activity in different cellular compartments to establish the physiological significance of trafficking defects.