ZNF268 Antibody, Biotin conjugated

What is ZNF268 and what is its biological significance?

ZNF268 is a typical KRAB-C2H2 zinc finger protein that plays important roles in hematopoiesis and has been implicated in leukemogenesis. The human ZNF268 gene encodes this protein, which functions as a transcription factor with potential regulatory effects on cell proliferation, apoptosis, and differentiation. Research has demonstrated that ZNF268 expression continuously decreases during erythropoiesis, suggesting it has stage-specific functions in blood cell development . Studies using K562 erythroleukemia cells have shown that knockdown of ZNF268 accelerates cellular proliferation, suppresses apoptosis, and reduces expression of erythroid-specific developmental markers, indicating its importance in maintaining the balance between proliferation and differentiation in hematopoietic cells .

How is ZNF268 expression regulated during erythropoiesis?

ZNF268 expression is transcriptionally downregulated during erythropoiesis through direct repression by GATA-1, a master regulator of erythropoiesis. Experimental evidence from electrophoretic mobility shift assays (EMSAs) and chromatin immunoprecipitation (ChIP) assays has confirmed that GATA-1 directly binds to a specific GATA binding site (G1 site, located at -1412 to -1388) in the ZNF268 promoter both in vitro and in vivo . This binding is associated with FOG (Friend of GATA), an interacting partner of GATA-1 that lacks sequence binding activity but contributes to GATA-1's regulatory functions. The repression of ZNF268 by GATA-1 appears to be crucial for normal erythroid development, as maintenance of ZNF268 expression inhibits differentiation and promotes proliferation in erythroleukemia cells .

What are the primary research applications for biotin-conjugated ZNF268 antibodies?

Biotin-conjugated ZNF268 antibodies serve multiple crucial research applications in molecular and cellular biology studies. These applications include:

• Chromatin immunoprecipitation (ChIP) assays to investigate protein-DNA interactions involving ZNF268

• Immunofluorescence microscopy for subcellular localization studies

• Flow cytometry for quantitative analysis of ZNF268 expression in cell populations

• Western blotting for protein expression analysis

• Immunoprecipitation to study protein-protein interactions

• Protein arrays to examine binding specificity

The biotin conjugation significantly enhances detection sensitivity through the strong biotin-streptavidin interaction, allowing for signal amplification and improved detection of low-abundance ZNF268 protein in experimental samples .

How does GATA-1 regulation of ZNF268 impact experimental design for studying erythroid differentiation?

When designing experiments to study erythroid differentiation using ZNF268 antibodies, researchers must account for the complex regulatory relationship between GATA-1 and ZNF268. The research data indicates that GATA-1 represses ZNF268 expression by directly binding to its promoter, specifically at the G1 binding site (-1412 to -1388) . This repression is functionally important, as ZNF268 silencing accelerates K562 cell proliferation, alters cell cycle distribution, and affects the expression of key regulatory proteins including c-myc, p53, and cyclin D1 .

Effective experimental design should include:

• Time-course analyses to capture the dynamic relationship between GATA-1 activity and ZNF268 expression

• Controls for GATA-1 expression and activity levels

• Assessment of FOG co-factor recruitment, as ChIP assays have shown positive signals with both anti-GATA-1 and anti-FOG antibodies

• Parallel analysis of downstream targets affected by ZNF268 silencing

A comprehensive approach would combine ChIP assays using biotin-conjugated ZNF268 antibodies with functional studies of cell proliferation, differentiation markers, and gene expression profiling to fully characterize the regulatory networks involved in erythroid development.

What are the optimal conditions for ZNF268 antibody validation in ChIP experiments?

Validating biotin-conjugated ZNF268 antibodies for ChIP experiments requires rigorous attention to experimental conditions. Based on the ChIP methodologies described in the research literature, optimal conditions include:

• Crosslinking: 1% formaldehyde at room temperature for 15 minutes provides effective crosslinking of protein-DNA complexes

• Sonication parameters: Optimize to generate chromatin fragments of 200-500 bp

• Antibody specificity controls:

  • Include isotype control antibodies (e.g., IgG) as negative controls

  • Use known target regions as positive controls

  • Include regions lacking ZNF268 binding sites as negative genomic controls

For primer design in ChIP-qPCR validation, follow the approach used in the reference study, where primers G1-s/G1-a (-1406 to -1266) were used to amplify the promoter region containing the G1 site, yielding a 141 bp fragment . Negative control primers should target regions without known ZNF268 binding sites, similar to the C-s/C-a (-1166 to -962) primers used in the reference study .

How can researchers resolve data contradictions in ZNF268 knockdown studies?

Resolving contradictory data in ZNF268 knockdown studies requires systematic troubleshooting and validation approaches. When conflicting results emerge, consider these methodological strategies:

• Validate knockdown efficiency at both mRNA and protein levels using complementary techniques (qRT-PCR and Western blotting with biotin-conjugated ZNF268 antibodies)

• Employ multiple siRNA/shRNA sequences to rule out off-target effects

• Implement rescue experiments by reintroducing ZNF268 expression to confirm phenotype specificity

• Analyze cell-type specific effects, as ZNF268 function may vary between different hematopoietic lineages

The reference study demonstrated successful ZNF268 knockdown validation by confirming reduced expression at both mRNA and protein levels compared to control lentiviral vector-infected cells . Their approach combined multiple functional assays (proliferation, cell cycle analysis, EdU incorporation) to comprehensively characterize the knockdown phenotype, providing a model for resolving apparently contradictory data through methodological thoroughness.

What is the recommended protocol for ChIP assays using biotin-conjugated ZNF268 antibodies?

Based on the successful ChIP methodologies described in the research literature, the recommended protocol for ChIP assays using biotin-conjugated ZNF268 antibodies follows these key steps:

• Crosslinking and Cell Preparation:

  • Crosslink cells with 1% formaldehyde at room temperature for 15 minutes

  • Wash cells twice with PBS

  • Lyse cells in sodium dodecyl sulfate lysis buffer

• Chromatin Preparation:

  • Prepare chromatin fragments by sonicating lysates on ice

  • Optimize sonication to generate fragments of 200-500 bp

• Immunoprecipitation:

  • Incubate lysates with biotin-conjugated ZNF268 antibody

  • Include appropriate controls (IgG, input samples)

  • Collect immunoprecipitated complexes using streptavidin-agarose beads

• Washing and Elution:

  • Wash pellets with dialysis buffer (2 mM EDTA and 50 mM Tris-HCl, pH 8.0)

  • Incubate at 65°C for 4 hours to reverse formaldehyde cross-links

  • Digest with 20 mg/ml proteinase K for 1 hour

• DNA Purification and Analysis:

  • Purify DNA using appropriate purification kit

  • Subject to PCR amplification using primers specific for regions of interest

  • Analyze by quantitative PCR or sequencing

For optimal results, incorporate the validation controls described in section 2.2 above and design primers similar to those listed in Table 1 of the reference study .

How should researchers troubleshoot weak or nonspecific signals when using ZNF268 antibodies?

When troubleshooting weak or nonspecific signals with biotin-conjugated ZNF268 antibodies, implement the following systematic approach:

• For Weak Signals:

  • Increase antibody concentration incrementally

  • Optimize incubation time and temperature

  • Enhance signal detection using amplification systems (e.g., avidin-biotin complex)

  • Reduce stringency of washing steps

  • Ensure target protein is not degraded during sample preparation

• For Nonspecific Signals:

  • Validate antibody specificity using ZNF268-silenced cells as negative controls

  • Increase blocking reagent concentration (BSA or non-fat dry milk)

  • Add detergents (0.1-0.5% Triton X-100) to reduce non-specific binding

  • Perform pre-clearing of lysates with protein A/G beads before immunoprecipitation

  • Increase stringency of washing steps

• Validation Controls:

  • Include competitive binding assays similar to those used in EMSA experiments with wild-type and mutant probes

  • Use multiple antibodies targeting different epitopes of ZNF268

  • Implement super-shift assays to confirm specificity

The reference study demonstrated effective validation of binding specificity in EMSA assays using competitive binding with unlabeled probes and mutant probes, which provides a model for antibody specificity validation .

What controls are essential when using biotin-conjugated ZNF268 antibodies for protein expression analysis?

When analyzing protein expression using biotin-conjugated ZNF268 antibodies, these essential controls ensure reliable and interpretable results:

• Positive Controls:

  • Cell lines with confirmed ZNF268 expression (e.g., K562 cells)

  • Recombinant ZNF268 protein standards for quantification

  • Previously validated sample preparations

• Negative Controls:

  • ZNF268-silenced cell lines (e.g., K562 cells transfected with ZNF268 shRNA)

  • Isotype control antibodies

  • Secondary antibody-only controls to assess non-specific binding

• Loading and Normalization Controls:

  • Housekeeping proteins (e.g., β-actin as used in the reference study)

  • Total protein staining for normalization

  • Spike-in standards for technical validation

• Specificity Controls:

  • Peptide competition assays

  • Multiple antibodies targeting different ZNF268 epitopes

  • Western blot migration at expected molecular weight

When analyzing and presenting data, always normalize ZNF268 expression to appropriate housekeeping controls, as demonstrated in the reference study where β-actin served as an internal control for Western blot analysis .

What are the optimal experimental conditions for detecting ZNF268 using flow cytometry?

For optimal detection of ZNF268 using biotin-conjugated antibodies in flow cytometry, implement these technical specifications:

• Sample Preparation:

  • Collect approximately 1×10^5 cells and wash with PBS containing 1% BSA and 0.1% sodium azide

  • Fix cells with 2-4% paraformaldehyde for 15 minutes at room temperature

  • Permeabilize with 0.1-0.5% Triton X-100 or 70% ethanol (overnight at 4°C for cell cycle analysis)

• Staining Protocol:

  • Block with 2-5% BSA or serum for 30 minutes

  • Incubate with biotin-conjugated ZNF268 antibody at optimized concentration

  • Wash thoroughly with PBS/BSA buffer

  • Incubate with fluorochrome-conjugated streptavidin

  • Include DNA staining if performing cell cycle analysis (e.g., propidium iodide)

• Instrument Settings:

  • Set appropriate voltage for fluorescence detection channels

  • Perform compensation if using multiple fluorochromes

  • Collect sufficient events (minimum 10,000) for statistical validity

• Analysis Approach:

  • Use appropriate gating strategies to eliminate debris and doublets

  • Analyze ZNF268 expression in relation to cell cycle phases if relevant

  • Compare with isotype controls to determine positive population thresholds

The reference study successfully applied flow cytometry for cell cycle and apoptosis analysis in ZNF268-silenced K562 cells, demonstrating that this technique can effectively detect biological consequences of ZNF268 regulation .

How can researchers optimize Western blot conditions for ZNF268 detection?

Optimizing Western blot conditions for ZNF268 detection requires attention to these key parameters:

• Sample Preparation:

  • Extract proteins using appropriate lysis buffer

  • Include protease inhibitors to prevent degradation

  • Quantify protein concentration for equal loading

  • Denature samples at 95°C for 5 minutes in sample buffer

• Gel Electrophoresis:

  • Use 8-10% SDS-PAGE gels for optimal resolution of ZNF268

  • Load 25-50 μg of total protein per lane

  • Include molecular weight markers

• Transfer Conditions:

  • Optimize transfer time and voltage for complete transfer

  • Use PVDF membrane for improved protein binding

  • Verify transfer efficiency with reversible protein staining

• Immunodetection:

  • Block membrane with 5% non-fat dry milk or BSA

  • Incubate with biotin-conjugated ZNF268 antibody (optimized dilution)

  • Wash thoroughly with TBST buffer

  • Detect using streptavidin-HRP and enhanced chemiluminescence

  • Include β-actin as loading control

• Quantification:

  • Use digital imaging systems for quantification

  • Normalize ZNF268 signal to loading control

  • Perform statistical analysis across multiple experiments

The reference study successfully employed Western blot to detect changes in ZNF268, c-myc, p53, and cyclin D1 expression following ZNF268 silencing, demonstrating the effectiveness of this approach for protein expression analysis .

How can biotin-conjugated ZNF268 antibodies be used to investigate protein-protein interactions?

Biotin-conjugated ZNF268 antibodies offer several advantages for investigating protein-protein interactions through these methodological approaches:

• Co-Immunoprecipitation (Co-IP):

  • Use biotin-conjugated ZNF268 antibodies to precipitate ZNF268 complexes

  • Capture with streptavidin beads for high-affinity binding

  • Analyze co-precipitated proteins by mass spectrometry or Western blot

  • Consider reversible crosslinking to capture transient interactions

• Proximity Ligation Assay (PLA):

  • Combine biotin-conjugated ZNF268 antibodies with antibodies against potential interacting partners

  • Visualize interactions through fluorescence microscopy

  • Quantify interaction signals to determine relative interaction strengths

• Bimolecular Fluorescence Complementation (BiFC):

  • Use ZNF268 antibodies to validate BiFC results

  • Confirm expression levels of fusion proteins

• Pull-down Assays:

  • Similar to the methodology used in the GATA-1/FOG studies in the reference paper

  • Use biotin-conjugated ZNF268 antibodies to validate interactions

The reference research demonstrated important protein interactions using ChIP assays with antibodies against GATA-1 and its interacting partner FOG. This approach revealed that both proteins associate with the ZNF268 promoter in vivo, suggesting a regulatory complex involving multiple proteins .

What are the future research directions for ZNF268 in hematopoietic disorders?

Future research directions for ZNF268 in hematopoietic disorders should build upon the foundational understanding established in the reference study, which demonstrated ZNF268's role in proliferation, apoptosis, and differentiation of K562 erythroleukemia cells . Key research directions include:

• Clinical Correlation Studies:

  • Examine ZNF268 expression patterns across various hematopoietic malignancies

  • Correlate expression levels with clinical outcomes and treatment responses

  • Develop biomarkers based on ZNF268 expression or mutation status

• Molecular Mechanism Investigations:

  • Expand on the finding that ZNF268 silencing affects c-myc, p53, and cyclin D1 expression

  • Map the complete gene regulatory network controlled by ZNF268

  • Identify direct transcriptional targets using ChIP-seq approaches

• Therapeutic Target Development:

  • Explore ZNF268 as a potential therapeutic target based on its role in cell proliferation

  • Develop compounds that modulate ZNF268 activity or expression

  • Test in xenograft models similar to those described in the reference study

• In Vivo Models:

  • Extend beyond the subcutaneous xenograft model described in the study

  • Develop conditional knockout models to study tissue-specific effects

  • Investigate ZNF268's role in normal hematopoiesis vs. leukemogenesis

The reference study demonstrated that ZNF268-silenced K562 cells promoted growth of subcutaneous tumors in nude mice, suggesting that targeting ZNF268 could have therapeutic relevance for certain hematopoietic disorders .