znf148 Antibody

What is ZNF148 and why is it studied in cancer research?

ZNF148, also known as Transcription factor ZBP-89 or Zinc finger DNA-binding protein 89, is a Kruppel-like zinc finger protein that functions as a transcriptional regulator. It represses the transcription of several genes including gastrin, stromelysin, and enolase by binding to G-rich boxes in their enhancer regions . ZNF148 has gained significant attention in cancer research because of its potential tumor suppressor role in colorectal cancer (CRC). Studies have demonstrated that ZNF148 expression patterns fluctuate during CRC development, increasing from normal mucosa to stage I CRC, then progressively decreasing from stage I to stage IV . Lower ZNF148 expression in tumors correlates significantly with lymph node metastases, advanced TNM disease stage, poor differentiation, higher recurrence rates, and worse survival outcomes . These findings suggest ZNF148 may serve as both a prognostic biomarker and therapeutic target in oncology research.

What are the key applications for ZNF148 antibodies in molecular biology research?

ZNF148 antibodies serve multiple research applications with varying methodological requirements:

When selecting an antibody for a specific application, researchers should verify reactivity with their target species and validation data for their particular application to ensure reliable results .

How should I design experiments to detect both ZNF148 isoforms using antibodies?

ZNF148 has two documented alternative splicing isoforms: ZNF148FL (containing complete 794 amino acids) and ZNF148ΔN (lacking the amino-terminal 129 amino acids) . To detect both isoforms:

• Antibody selection: Choose antibodies targeting C-terminal regions (e.g., AA 690-794) to detect both isoforms . Antibodies against N-terminal regions will only detect the full-length variant.

• Western blot optimization: Expect two molecular weight bands - approximately 103 kDa for ZNF148FL and a lower molecular weight band for ZNF148ΔN . Use a gradient gel (4-12%) to achieve optimal separation.

• Controls: Include positive controls (e.g., HCT 116 or HepG2 cells) which express detectable levels of ZNF148 .

• Loading control: Normalize expression to housekeeping proteins (β-actin, GAPDH) for accurate quantification.

• Validation approach: For critical experiments, use two different antibodies targeting distinct epitopes to confirm isoform-specific detection .

This comprehensive approach enables reliable detection and differentiation of ZNF148 isoforms, which may have distinct functional roles in different biological contexts.

What are the optimal sample preparation methods for ZNF148 detection in different tissue types?

Sample preparation significantly impacts ZNF148 antibody performance across different tissue types:

For Western blotting:

• Extract proteins using RIPA or NP-40 buffer supplemented with protease inhibitors

• Include phosphatase inhibitors if studying phosphorylation status

• Sonicate samples briefly to shear DNA and reduce viscosity

• Heat samples at 95°C for 5 minutes in Laemmli buffer with β-mercaptoethanol

For Immunohistochemistry:

• Fixation in 10% neutral buffered formalin for 24-48 hours is optimal

• For antigen retrieval, use citrate buffer (pH 6.0) with heat-induced epitope retrieval

• Block endogenous peroxidase activity with 3% hydrogen peroxide

• Include a protein blocking step to reduce non-specific binding

For primary cells and culture models:

• LHCN-M2 and HSMM cells have been successfully used to study ZNF148 in muscle differentiation

• HCT 116 cells serve as positive controls for ZNF148 expression in colorectal cancer studies

• HeLa, 293T, and NIH3T3 cells also express detectable levels of ZNF148

These optimized protocols ensure consistent, reproducible results when working with ZNF148 antibodies across various experimental systems.

How can ZNF148 antibodies be utilized to study its role in colorectal cancer progression?

ZNF148 exhibits a unique expression pattern during colorectal cancer progression that can be studied using a multi-faceted antibody-based approach:

• Tissue microarray analysis: Employ immunohistochemistry with validated ZNF148 antibodies to analyze expression across different stages of colorectal cancer progression . This approach has revealed that ZNF148 expression initially increases from normal mucosa to stage I CRC, then progressively decreases from stage I to stage IV .

• Comparative analysis workflow:

  • Collect matched samples of normal mucosa, adenoma, primary tumor, and metastatic lesions

  • Perform IHC staining with standardized protocols and scoring systems

  • Correlate expression patterns with clinicopathological features

  • Analyze relationship to patient outcomes using Kaplan-Meier survival curves

• Mechanistic studies: Combine ZNF148 antibodies with antibodies against established colorectal cancer markers to investigate potential regulatory relationships. Consider dual immunofluorescence to visualize co-expression patterns.

• Validation in familial adenomatous polyposis models: Previous research has demonstrated that ZNF148 expression is upregulated during carcinogenesis in familial adenomatous polyposis , providing a model system for studying the transition from pre-malignant to malignant lesions.

This comprehensive approach can reveal insights into how ZNF148 contributes to colorectal cancer initiation, progression, and metastasis, potentially identifying new therapeutic targets or prognostic markers.

What are the methodological considerations for studying ZNF148's role in muscle differentiation using specific antibodies?

Investigating ZNF148's function as a negative regulator of muscle differentiation requires careful experimental design:

• Cell model selection:

  • LHCN-M2 immortalized human myoblasts and primary Human Skeletal Muscle Myoblasts (HSMM) have been validated for ZNF148 studies

  • Both cell types demonstrate robust myosin heavy chain (MHC) expression following ZNF148 knockdown

• Experimental knockdown approach:

  • siRNA transfection targeting ZNF148 effectively reduces expression

  • Confirm knockdown efficiency via western blot using validated antibodies

  • Monitor muscle differentiation markers (MHC, myogenin) by immunofluorescence and qPCR

• Time-course considerations:

  • ZNF148 levels remain relatively unchanged during normal LHCN-M2 differentiation

  • Following ZNF148 knockdown, monitor expression at 24h, 48h, and 72h timepoints

  • Assess both early (MYOD, MEF2C) and late (MHC) differentiation markers

• Flow cytometry protocol:

  • Fix cells in 2% paraformaldehyde

  • Permeabilize with 0.1% Triton X-100

  • Stain with anti-MHC antibody followed by fluorophore-conjugated secondary antibody

  • Use appropriate gating strategy to quantify MHC-positive cells

• Controls and validation:

  • Include non-targeting siRNA controls

  • Perform rescue experiments with ZNF148 overexpression

  • Validate findings with multiple ZNF148 antibodies targeting different epitopes

This methodological framework enables robust analysis of ZNF148's regulatory role in muscle differentiation while minimizing experimental artifacts.

How can researchers address variability in ZNF148 antibody performance across different experimental systems?

Inconsistent antibody performance can significantly impact ZNF148 research. Address these challenges with a systematic approach:

• Antibody validation strategy:

  • Confirm specificity using blocking peptides - as demonstrated with ab69933, where peptide competition eliminated the 89 kDa band in HepG2 extracts

  • Validate results with at least two antibodies targeting different epitopes

  • Include positive control samples with known ZNF148 expression (HCT 116, HepG2, HeLa cells)

• Sample-specific optimization:

  • For each new cell line or tissue type, perform antibody titration (1:500 to 1:4000 for WB)

  • Test multiple extraction methods (RIPA vs. NP-40 buffers)

  • Optimize blocking conditions (BSA vs. non-fat dry milk)

• Species cross-reactivity considerations:

  • Some antibodies show reactivity with human, mouse and rat samples

  • Others are human-specific

  • Always verify species reactivity before cross-species comparisons

• Addressing multiple bands:

  • ZNF148 can appear at both 89 kDa and 95-103 kDa due to alternative splicing

  • Post-translational modifications may cause additional bands

  • Document all observed bands and validate with appropriate controls

• Storage and handling:

  • Store antibodies according to manufacturer recommendations (-20°C with glycerol)

  • Avoid repeated freeze-thaw cycles

  • For long-term storage, aliquot antibodies to maintain optimal performance

By implementing these strategies, researchers can achieve consistent, reproducible results when working with ZNF148 antibodies across diverse experimental systems.

What challenges exist in interpreting ZNF148 expression data in cancer studies, and how can they be addressed?

Interpreting ZNF148 expression in cancer contexts presents several methodological challenges:

How are ZNF148 antibodies being applied to investigate its potential role as a biomarker in colorectal cancer?

ZNF148's complex expression pattern during colorectal cancer progression makes it a promising biomarker candidate, with several active research directions:

These research directions highlight how ZNF148 antibodies are contributing to the development of more personalized approaches to colorectal cancer management, potentially improving patient stratification and treatment selection.

What methodological approaches are being developed to study ZNF148's interaction with other transcription factors?

Investigating ZNF148's complex interactions with other transcription factors requires sophisticated methodological approaches:

• Co-immunoprecipitation (Co-IP) strategies:

  • Use ZNF148 antibodies to pull down protein complexes from nuclear extracts

  • Analyze interacting partners by mass spectrometry

  • Confirm specific interactions with reciprocal Co-IP experiments

  • Include RNase treatment to distinguish RNA-dependent from direct protein-protein interactions

• Chromatin immunoprecipitation (ChIP) methods:

  • Perform ChIP with anti-ZNF148 antibodies to identify genomic binding sites

  • Develop sequential ChIP (ChIP-reChIP) protocols to identify co-binding with other factors

  • Combine with high-throughput sequencing (ChIP-seq) for genome-wide binding profiles

• Proximity ligation assays (PLA):

  • Visualize and quantify protein-protein interactions in situ

  • Requires careful antibody selection to ensure compatible species and epitope accessibility

  • Protocols must be optimized for each tissue or cell type

• CRISPR-based approaches:

  • Generate tagged endogenous ZNF148 for real-time interaction studies

  • Create domain-specific mutations to map interaction interfaces

  • Combine with rapid immunoprecipitation mass spectrometry (RIME) for comprehensive interactome analysis

• Functional interaction studies:

  • Luciferase reporter assays to measure transcriptional effects of ZNF148 with potential partners

  • Electrophoretic mobility shift assays (EMSA) using purified factors and target DNA sequences

  • Nuclear co-localization studies using fluorescently-tagged factors

These methodological approaches provide complementary insights into how ZNF148 functions within transcriptional complexes, potentially revealing new therapeutic targets in diseases where ZNF148 dysregulation contributes to pathogenesis.

What quality control measures are essential when validating a new ZNF148 antibody for research applications?

Rigorous validation is critical before implementing a new ZNF148 antibody in research protocols:

• Specificity verification:

  • Peptide competition assays using the immunizing peptide should eliminate specific signal

  • siRNA/shRNA knockdown should reduce antibody signal proportionally to protein reduction

  • Test in multiple cell lines with known ZNF148 expression (HCT 116, HepG2, HeLa)

  • Verify appropriate molecular weight (89-103 kDa depending on isoform)

• Application-specific validation:

  • For Western blot: Test linearity of detection across a dilution series (5-50 µg protein)

  • For IHC: Compare staining in normal vs. tumoral tissue, with appropriate controls

  • For IF: Confirm expected subcellular localization (primarily nuclear)

• Epitope mapping considerations:

  • Document the specific region targeted (N-terminal vs. C-terminal)

  • Antibodies targeting AA 690-794 will detect both major isoforms

  • N-terminal antibodies (AA 106-135) will only detect full-length protein

• Cross-reactivity assessment:

  • Test across target species (human, mouse, rat) if multi-species applications are planned

  • Verify lack of signal in ZNF148 knockout models when available

• Batch consistency:

  • Maintain reference samples to test each new antibody lot

  • Document lot-to-lot variations in sensitivity and background

These validation steps ensure that experimental findings with ZNF148 antibodies are reliable, reproducible, and biologically relevant, reducing the risk of artifacts or misinterpretations in research outcomes.

How should researchers approach contradictory data when comparing ZNF148 expression results from different antibodies or platforms?

When faced with conflicting ZNF148 expression data, a systematic troubleshooting approach is essential:

• Epitope-specific considerations:

  • Different antibodies target distinct regions of ZNF148, potentially recognizing different isoforms

  • Map the epitopes of each antibody relative to known functional domains and splice variants

  • N-terminal vs. C-terminal antibodies may yield different results due to isoform specificity

• Methodological reconciliation:

  • Compare detection methods (WB vs. IHC vs. IF) and extraction protocols

  • Evaluate antibody dilutions, incubation conditions, and detection systems

  • Standardize protein loading and normalization approaches

• Biological context assessment:

  • ZNF148 expression varies by tissue type, developmental stage, and disease progression

  • Expression increases from normal mucosa to stage I colorectal cancer, then decreases through later stages

  • Document precise origin of samples, including stage and grade for cancer tissues

• Technical validation experiments:

  • Perform side-by-side comparisons with multiple antibodies on identical samples

  • Include positive controls (HCT 116, HepG2 cells) and negative controls (siRNA knockdown)

  • Use orthogonal methods (protein vs. mRNA detection) to corroborate findings

• Integrated data analysis:

  • Weight findings based on validation quality and consistency across experiments

  • Consider meta-analysis approaches when multiple datasets are available

  • Acknowledge limitations and contradictions transparently in publications