kcmf1 Antibody

What is KCMF1 and why is it significant for research?

KCMF1 (Potassium Channel Modulatory Factor 1) is a 381-amino acid protein with intrinsic E3 ubiquitin ligase activity that plays a crucial role in cellular processes by facilitating ubiquitination of target proteins. This post-translational modification regulates protein degradation and function, maintaining cellular homeostasis and influencing various signaling pathways. KCMF1 contains one C2H2-type zinc finger and one ZZ-type zinc finger domain, which are critical for DNA and protein interactions, enabling its role in transcriptional regulation . Research significance includes its upregulation by basic fibroblast growth factor (FGF-2) in gastric cancer cells and its involvement in epithelial morphogenesis . Moreover, KCMF1 is part of a ubiquitin ligase complex with UBA6, BIRC6, and UBR4, which is required for the survival of a subset of epithelial tumors .

What are the main types of KCMF1 antibodies available for research?

KCMF1 antibodies are available in several formats based on host species, clonality, and target epitopes:

These antibodies are available in various conjugated forms including HRP, FITC, PE, and biotinylated formats for specialized detection methods .

How should researchers select the appropriate KCMF1 antibody for their experiments?

Selection of the appropriate KCMF1 antibody should be guided by several experimental considerations:

• Application compatibility: Verify that the antibody has been validated for your specific application (WB, IF, IHC, IP, ELISA) .

• Species reactivity: Ensure the antibody recognizes KCMF1 in your experimental species. Some antibodies have broad cross-reactivity (human, mouse, rat, cow, etc.) .

• Epitope location: Consider whether targeting a specific domain (C-terminal, N-terminal, or internal epitope) is relevant to your research question, especially if studying specific isoforms or post-translational modifications .

• Clonality requirements: Choose monoclonal antibodies for higher specificity and consistency across experiments, or polyclonal antibodies for enhanced sensitivity and recognition of multiple epitopes .

• Detection methodology: Select unconjugated antibodies if using secondary detection systems, or appropriate conjugates (HRP, fluorescent dyes) if direct detection is preferred .

Validation data from the manufacturer and literature citations should be reviewed before making a final selection.

What are the optimal storage and handling conditions for KCMF1 antibodies?

While specific storage recommendations may vary slightly between manufacturers, the following general guidelines should be followed to maintain antibody integrity and performance:

• Storage temperature: Most KCMF1 antibodies should be stored at -20°C for long-term storage, with aliquots at 4°C for ongoing experiments.

• Aliquoting: Upon receipt, divide the antibody into small, single-use aliquots to minimize freeze-thaw cycles, which can degrade protein structure and reduce activity.

• Working dilutions: Prepare working dilutions immediately before use and avoid storing diluted antibodies for extended periods.

• Buffer compatibility: Some antibodies may contain preservatives like sodium azide, which can inhibit HRP activity in certain applications—verify compatibility with your downstream assays.

• Shipping conditions: Note that some KCMF1 antibodies, particularly monoclonal formulations, may be shipped on dry ice and require immediate storage at recommended temperatures upon receipt .

For conjugated antibodies (FITC, HRP, etc.), protect from light to prevent photobleaching of fluorophores.

How can KCMF1 antibodies be optimized for studying protein-protein interactions in the ubiquitin pathway?

Optimizing KCMF1 antibodies for protein-protein interaction studies requires strategic experimental design:

• Co-immunoprecipitation (Co-IP): Select antibodies targeting different epitopes from potential interaction partners. The A-3 mouse monoclonal antibody has been specifically validated for immunoprecipitation studies . When investigating KCMF1's interactions with UBA6, BIRC6, or UBR4, use gentle lysis buffers (containing 0.5-1% NP-40 or Triton X-100) to preserve protein complexes.

• Proximity ligation assays (PLA): When using mouse monoclonal KCMF1 antibodies, pair them with rabbit polyclonal antibodies against suspected interaction partners for optimal PLA results.

• FRET/BRET approaches: For live-cell interaction studies, epitope accessibility must be considered—C-terminal targeting antibodies may be preferred as the C-terminal region contains important regulatory domains for protein interactions .

• Crosslinking strategies: Prior to immunoprecipitation with KCMF1 antibodies, use membrane-permeable crosslinkers like DSP (dithiobis[succinimidyl propionate]) to stabilize transient interactions within the ubiquitin pathway.

• Native PAGE analysis: Use non-denaturing conditions with appropriate KCMF1 antibodies to detect intact protein complexes.

Research has demonstrated that endogenous BIRC6 binds to both UBA6 and KCMF1, as detected through protein complex isolation using anti-FLAG antibodies . This approach can be adapted for studying novel KCMF1 interactions by using the appropriate KCMF1 antibody.

What are the key considerations for using KCMF1 antibodies in cancer research models?

When employing KCMF1 antibodies in cancer research, several critical considerations should guide experimental design:

How should researchers troubleshoot non-specific binding when using KCMF1 antibodies?

Non-specific binding is a common challenge when working with antibodies. For KCMF1 antibodies, consider these troubleshooting approaches:

• Antibody validation: Verify antibody specificity using positive and negative controls. For human samples, compare KCMF1 expression in tissues known to have high (spleen, small intestine) versus low expression (brain, liver) .

• Blocking optimization: Test different blocking agents (5% BSA, 5% non-fat milk, commercial blockers) to reduce background. For phospho-rich samples, BSA may be preferable over milk proteins which contain phosphoproteins.

• Antibody titration: Perform dilution series experiments to determine optimal antibody concentration that maximizes specific signal while minimizing background. Start with the manufacturer's recommended dilution and test 2-fold dilutions in both directions.

• Washing stringency adjustment: Increase washing steps or detergent concentration (0.1% to 0.3% Tween-20) in buffer to reduce non-specific binding, especially when using polyclonal antibodies targeting the C-terminal region .

• Cross-adsorption: If cross-reactivity with related proteins is suspected, pre-incubate the antibody with the purified cross-reactive protein or consider switching to a monoclonal antibody like A-3 which targets a more specific epitope.

• Epitope competition: Confirm specificity by peptide competition assays using the immunogen peptide (e.g., the synthetic peptide from amino acids 280-308 for C-terminal targeting antibodies) .

• Alternative detection methods: If problems persist with one application (e.g., IHC), try an alternative method like Western blotting where size discrimination can help confirm specificity.

What are the considerations for using KCMF1 antibodies in co-localization studies with ubiquitination machinery?

Successful co-localization studies examining KCMF1's interaction with ubiquitination machinery require careful methodological planning:

• Antibody compatibility: When performing double immunostaining, select KCMF1 antibodies raised in different host species than antibodies targeting other ubiquitination components (UBA6, BIRC6, UBR4) to allow simultaneous detection .

• Fixation optimization: Test multiple fixation methods (4% paraformaldehyde, methanol, or acetone) as fixation can affect epitope accessibility, particularly for conformationally sensitive protein complexes in the ubiquitination pathway.

• Signal amplification strategies: For low-abundance interactions, consider tyramide signal amplification (TSA) with HRP-conjugated KCMF1 antibodies to enhance detection sensitivity .

• Super-resolution microscopy: Standard confocal microscopy may not resolve closely associated proteins within ubiquitination complexes. Techniques like STORM or STED microscopy with appropriately conjugated KCMF1 antibodies (e.g., Alexa Fluor conjugates) can provide nanometer-scale resolution .

• Live-cell imaging considerations: For dynamic studies of KCMF1 trafficking with ubiquitination machinery, cell-permeable antibody fragments or expression of tagged constructs may be necessary, as conventional antibodies cannot access intracellular targets in living cells.

• Quantitative co-localization analysis: Employ Pearson's correlation coefficient or Manders' overlap coefficient to quantify the degree of co-localization between KCMF1 and ubiquitination machinery components, rather than relying on qualitative assessment alone.

• Controls for specificity: Include appropriate controls such as cells lacking KCMF1 expression or pre-absorption of the antibody with recombinant KCMF1 protein to confirm staining specificity.

How can researchers validate KCMF1 antibody specificity for their particular experimental system?

Comprehensive validation of KCMF1 antibody specificity should involve multiple complementary approaches:

• Western blot analysis: Confirm detection of a band at the expected molecular weight of KCMF1 (approximately 42 kDa). Polyclonal antibodies targeting the C-terminal region (AA 280-308) and monoclonal antibodies like A-3 have been validated for Western blotting applications .

• Knockdown/knockout controls: Compare antibody signal in wild-type cells versus those with KCMF1 knockdown (siRNA/shRNA) or knockout (CRISPR-Cas9). Complete signal elimination in knockout samples provides strong evidence of specificity.

• Overexpression controls: Transfect cells with tagged KCMF1 constructs and confirm co-localization of antibody signal with the tag-specific signal.

• Peptide competition: Pre-incubate the antibody with the immunizing peptide before application to samples. Signal reduction indicates specific binding to the target epitope.

• Cross-species reactivity assessment: Test the antibody on samples from different species when working with non-human models. For example, certain KCMF1 antibodies show reactivity with human, mouse, rat, and other species .

• Multiple antibody concordance: Compare staining patterns using antibodies targeting different KCMF1 epitopes (e.g., N-terminal vs. C-terminal). Similar patterns increase confidence in specificity.

• Mass spectrometry validation: Perform immunoprecipitation with the KCMF1 antibody followed by mass spectrometry to confirm the identity of the precipitated protein.

• Immunohistochemical distribution: Compare the tissue distribution pattern detected by the antibody with known KCMF1 expression profiles (higher in spleen, small intestine, ovary, etc.) .

What are the optimal sample preparation methods for detecting KCMF1 in different experimental contexts?

Sample preparation requirements vary by application and tissue type when detecting KCMF1:

• Western blotting:

  • Cell lysis: RIPA buffer supplemented with protease inhibitors is generally effective. For detecting KCMF1 interactions with ubiquitination machinery, milder NP-40 or Triton X-100 based buffers may better preserve protein complexes .

  • Protein denaturation: Standard Laemmli buffer with DTT or β-mercaptoethanol, heated at 95°C for 5 minutes.

  • Gel percentage: 10-12% polyacrylamide gels typically provide optimal resolution for the 42 kDa KCMF1 protein.

• Immunohistochemistry/Immunofluorescence:

  • Fixation: 4% paraformaldehyde (10-15 minutes) typically preserves KCMF1 antigenicity while maintaining cellular architecture.

  • Antigen retrieval: Heat-induced epitope retrieval in citrate buffer (pH 6.0) is recommended for most KCMF1 antibodies, particularly for formalin-fixed tissues.

  • Permeabilization: 0.1-0.3% Triton X-100 for 10 minutes allows antibody access to intracellular KCMF1.

  • Blocking: 5% normal serum from the species of the secondary antibody, 1 hour at room temperature.

• Immunoprecipitation:

  • Cell lysis: Gentle lysis in 1% NP-40 buffer with 150mM NaCl preserves KCMF1 protein complexes.

  • Pre-clearing: Pre-clear lysates with protein A/G beads to reduce non-specific binding.

  • Antibody incubation: Overnight at 4°C with gentle rotation for optimal KCMF1 capture.

  • Washing: Multiple washes with decreasing salt concentration to maintain specific interactions.

• Flow cytometry:

  • Fixation: 2% paraformaldehyde for 10-15 minutes.

  • Permeabilization: 0.1% saponin or 0.1% Triton X-100 for intracellular KCMF1 detection.

  • Antibody incubation: Use fluorophore-conjugated KCMF1 antibodies or appropriate secondary antibodies.

How can researchers optimize KCMF1 antibody concentrations for different applications?

Optimization of KCMF1 antibody concentrations is critical for generating reliable, reproducible results across different applications:

• Western blotting optimization:

  • Starting concentration: Begin with the manufacturer's recommended dilution (typically 1:1000 for most KCMF1 antibodies) .

  • Titration approach: Prepare a mini dilution series (e.g., 1:500, 1:1000, 1:2000, 1:5000) using identical sample loads.

  • Evaluation criteria: Select the dilution that provides the strongest specific band at 42 kDa with minimal background.

  • Exposure optimization: When using chemiluminescence detection, capture multiple exposure times to determine the optimal signal-to-noise ratio.

• Immunohistochemistry/Immunofluorescence optimization:

  • Starting concentration: Begin with 1-5 μg/ml for monoclonal antibodies like A-3 or 7G2C11G2 .

  • Positive control tissues: Use tissues known to express KCMF1 (spleen, small intestine) for initial optimization .

  • Signal amplification considerations: If using tyramide signal amplification, reduce primary antibody concentration by 5-10 fold from standard protocols.

  • Incubation conditions: Test both overnight 4°C and 1-2 hour room temperature incubations to determine optimal conditions.

• Flow cytometry optimization:

  • Starting concentration: Begin with 1 μg per million cells for direct conjugates or 0.5-1 μg for unconjugated antibodies.

  • Titration method: Set up a geometric dilution series and identify the saturation point where signal plateaus.

  • Controls: Include fluorescence-minus-one (FMO) controls to accurately set gates and determine positive staining.

• ELISA optimization:

  • Capture antibody: If using KCMF1 antibody as capture, test 1-10 μg/ml coating concentrations.

  • Detection antibody: For detection, begin with a 1:1000 dilution for enzyme-conjugated antibodies and adjust based on signal intensity.

  • Standard curve generation: Establish a standard curve using recombinant KCMF1 protein at 0.1-1000 ng/ml.

• Documentation and standardization:

  • Record all optimization parameters including antibody lot, dilution, incubation time/temperature, and detection method.

  • Once optimized, maintain consistent conditions across experiments for reproducibility.

How are KCMF1 antibodies being used to understand stress response pathways in cancer?

KCMF1 antibodies are becoming instrumental in elucidating the integrated stress response (ISR) mechanisms in cancer:

• Ubiquitination cascade characterization: Recent research has employed KCMF1 antibodies to identify its participation in a ubiquitin ligase complex with UBA6, BIRC6, and UBR4, which regulates the integrated stress response in epithelial tumors . Immunoprecipitation studies using anti-FLAG antibodies have demonstrated that endogenous BIRC6 binds to both UBA6 and KCMF1, suggesting a coordinated mechanism for stress adaptation .

• Epithelial cancer vulnerability mapping: KCMF1 antibodies are being used to profile protein expression across tumor types to identify cancers where the KCMF1-containing ubiquitin ligase complex represents a therapeutic vulnerability. These studies help identify patient populations where targeting this pathway might be most effective.

• Post-translational modification networks: Researchers are employing KCMF1 antibodies in combination with ubiquitin-specific antibodies to map the dynamic changes in protein ubiquitination during stress responses. This approach illuminates how cancer cells adapt to therapeutic interventions through modulation of protein stability.

• Therapeutic response prediction: KCMF1 expression and localization patterns detected by specific antibodies are being investigated as potential biomarkers for predicting tumor response to therapies targeting stress response pathways.

• Co-localization with stress granules: During integrated stress response activation, immunofluorescence studies with KCMF1 antibodies are revealing potential associations with stress granules, cytoplasmic RNA-protein complexes that form during cellular stress.

• Signaling pathway cross-talk: KCMF1 antibodies are facilitating studies of how the ubiquitination cascade intersects with other cancer-relevant pathways, particularly in relation to FGF-2 signaling in gastric cancer where KCMF1 is upregulated .

What are the emerging applications of KCMF1 antibodies in studying epithelial morphogenesis?

Emerging applications of KCMF1 antibodies in epithelial morphogenesis research include:

• Developmental timing studies: Immunohistochemistry with KCMF1 antibodies is enabling researchers to track temporal expression patterns during epithelial tissue formation in developmental models, revealing critical windows where KCMF1 function may be essential.

• 3D organoid research: KCMF1 antibodies are being applied to investigate protein localization in three-dimensional epithelial organoid cultures, providing insights into how KCMF1 contributes to complex tissue architecture formation that cannot be observed in traditional 2D cultures.

• Cell polarity regulation: Immunofluorescence studies using KCMF1 antibodies in conjunction with polarity markers are uncovering potential roles in establishing and maintaining epithelial cell polarity, a fundamental aspect of functional epithelial tissues.

• Epithelial-mesenchymal transition (EMT) investigation: Researchers are utilizing KCMF1 antibodies to track protein expression changes during EMT, a process critical in both development and cancer metastasis.

• Growth factor response mapping: Given KCMF1's upregulation by FGF-2 in gastric cancer cells , antibodies are being employed to elucidate the spatiotemporal dynamics of KCMF1 expression in response to various growth factors in epithelial models.

• Junction complex association studies: Co-immunoprecipitation with KCMF1 antibodies is revealing potential interactions with epithelial junction proteins, suggesting mechanisms by which KCMF1 might influence epithelial barrier function and intercellular communication.

• Wound healing models: KCMF1 antibodies are being applied in epithelial wound healing assays to understand protein dynamics during regenerative processes, with potential implications for therapeutic approaches to enhance tissue repair.

• Tissue-specific function analysis: Comparative studies using KCMF1 antibodies across different epithelial tissues (kidney, lung, intestine) are identifying tissue-specific functions and regulatory mechanisms that may explain the differential expression patterns observed .

What technological advancements are improving KCMF1 antibody applications in research?

Recent technological advancements are significantly enhancing the utility of KCMF1 antibodies in research:

• Super-resolution microscopy compatibility: Newer KCMF1 antibody formulations conjugated to photoactivatable fluorophores enable nanoscale visualization of protein localization and interactions using techniques like STORM, PALM, and STED microscopy, revealing previously undetectable spatial relationships within ubiquitination complexes.

• Multiplexed immunodetection systems: Advanced multiplexing platforms allow simultaneous detection of KCMF1 along with dozens of other proteins in a single sample, facilitating comprehensive pathway analysis. These techniques include:

  • Cyclic immunofluorescence (CycIF)

  • Mass cytometry (CyTOF)

  • Digital spatial profiling (DSP)

• Single-cell protein analysis: Integration of KCMF1 antibodies into single-cell proteomic workflows enables correlation of KCMF1 expression with cellular phenotypes at unprecedented resolution, revealing heterogeneity within seemingly homogeneous populations.

• Proximity labeling approaches: Combining KCMF1 antibodies with enzyme-mediated proximity labeling methods (BioID, APEX) allows identification of transient interaction partners that might be missed by traditional co-immunoprecipitation approaches.

• In vivo imaging capabilities: Development of near-infrared fluorophore-conjugated KCMF1 antibodies or antibody fragments enables non-invasive tracking of KCMF1 expression in living organisms using whole-animal imaging systems.

• Automated quantitative analysis: Integration of KCMF1 immunostaining with artificial intelligence-based image analysis platforms enables unbiased, high-throughput quantification of expression patterns across large sample cohorts.

• Antibody engineering advances: Recombinant antibody technology is producing KCMF1-specific single-chain variable fragments (scFvs) and nanobodies with enhanced tissue penetration and reduced background, particularly valuable for dense tissues where standard antibodies perform suboptimally.

• Spatially-resolved proteomics: Emerging techniques combining immunodetection with mass spectrometry imaging allow visualization of KCMF1 distribution while simultaneously mapping post-translational modifications and interaction partners with spatial context preserved.

These technological advancements are collectively transforming the landscape of KCMF1 research, enabling more precise characterization of its diverse roles in normal physiology and disease pathogenesis.

What are the key considerations for validating KCMF1 antibody results for publication?

When preparing KCMF1 antibody results for publication, researchers should address these critical validation requirements:

• Antibody characterization documentation: Provide complete details about the KCMF1 antibody used, including supplier, catalog number, clone designation (for monoclonals like A-3 or 7G2C11G2), lot number, and target epitope .

• Specificity validation evidence: Include controls demonstrating antibody specificity, such as:

  • KCMF1 knockdown/knockout validation

  • Peptide competition results

  • Comparative results with antibodies targeting different KCMF1 epitopes

  • Western blot showing a single band at the expected 42 kDa size

• Optimization documentation: Describe antibody dilutions, incubation conditions, and detection methods that were optimized for each experimental system and application.

• Reproducibility demonstration: Present data from biological replicates and technical replicates to demonstrate result consistency.

• Proper image acquisition parameters: Document microscopy settings (exposure times, gain, laser power) and image processing methods to ensure transparent and reproducible visualization of KCMF1.

• Quantification methodology: Clearly describe quantification methods for immunoblots, immunostaining, or other applications, including software used and statistical approaches.

• Tissue/cell line authentication: Confirm the identity of experimental systems where KCMF1 is being studied, especially when making claims about tissue-specific expression patterns .

• Cross-reactivity assessment: When studying KCMF1 in non-human systems, provide evidence that the antibody recognizes the target species ortholog as claimed .

• Batch effects consideration: Acknowledge potential batch-to-batch variability in antibody performance and describe how this was controlled for in experimental design.

• Data availability: Consider depositing raw image data in appropriate repositories to enhance transparency and enable reanalysis by other researchers.