ZNF253 Antibody

What is ZNF253 and why is it significant in research?

ZNF253, also named as Zinc finger protein 411 or BMZF1, is a 499 amino acid protein belonging to the krueppel C2H2-type zinc-finger protein family. It contains one KRAB domain and eleven C2H2-type zinc fingers, suggesting its function as a transcription factor . The protein is primarily expressed in bone marrow and in monocytic and immature erythroid cell lines, indicating potential roles in hematopoiesis and cell differentiation . While less studied than some other zinc finger proteins, emerging research suggests it may play important regulatory roles in cellular processes, making it a target of interest for researchers investigating transcriptional regulation mechanisms.

What are the optimal storage conditions for ZNF253 antibodies?

ZNF253 antibodies should be stored at -20°C for optimal stability and performance. Under these conditions, they remain stable for approximately one year after shipment . For the commercially available preparations, they are typically supplied in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 . It is important to note that repeated freeze-thaw cycles should be avoided to maintain antibody integrity and activity. For smaller volume antibodies (20μl sizes), some preparations may contain 0.1% BSA as a stabilizer . While aliquoting is a common practice for antibodies, some manufacturers specifically note that it is unnecessary for their -20°C storage formulations .

What applications are ZNF253 antibodies validated for?

Commercial ZNF253 antibodies have been validated primarily for Western Blot (WB) and ELISA applications . For Western blotting, the recommended dilution ranges from 1:200 to 1:1000, though optimal dilution should be determined for each experimental system . Positive Western blot results have been specifically documented in HepG2 cells and SMMC-7721 cells . While not as extensively documented, some antibodies may also be suitable for immunohistochemistry (IHC) applications, as suggested by studies using related zinc finger protein antibodies in tissue sections . It's important to note that sample-dependent optimization is recommended, and researchers should check validation data galleries provided by manufacturers for application-specific information .

What is the expected molecular weight for ZNF253 in Western blot applications?

When performing Western blot with ZNF253 antibodies, researchers should expect to observe bands in the range of 58-65 kDa . The calculated molecular weight based on the 499 amino acid sequence is approximately 58 kDa, but post-translational modifications or other factors may account for the slightly higher observed molecular weights in some experimental conditions . This variation in detected molecular weight is common for many proteins and can be influenced by the cell or tissue type being analyzed, sample preparation methods, and the specific antibody being used. When troubleshooting unexpected results, researchers should consider running positive controls such as lysates from HepG2 or SMMC-7721 cells, which have been documented to express detectable levels of ZNF253 .

How can I design experiments to investigate ZNF253's role as a transcription factor?

To investigate ZNF253's role as a transcription factor, consider implementing a multi-faceted experimental approach. Begin with chromatin immunoprecipitation (ChIP) assays using validated ZNF253 antibodies to identify genomic binding sites. Follow with reporter gene assays incorporating putative target promoters to assess transcriptional activation or repression capabilities. The presence of a KRAB domain suggests potential repressor activity . For functional validation, employ gain-of-function and loss-of-function studies using overexpression constructs and siRNA/shRNA approaches similar to those designed for related zinc finger proteins . The siRNA/shRNA design could follow patterns used for other zinc finger proteins; for example, sequences targeting the unique regions outside the conserved zinc finger domains . Additionally, co-immunoprecipitation experiments can identify protein interaction partners that may form part of ZNF253's transcriptional regulatory complex. Analysis of gene expression changes following ZNF253 manipulation can be performed through RNA-seq or qRT-PCR of candidate target genes.

How should I address cross-reactivity concerns when using ZNF253 antibodies in systems with multiple zinc finger proteins?

Addressing cross-reactivity concerns requires careful experimental design when studying ZNF253 in systems expressing multiple zinc finger proteins. First, perform extensive validation through comparative analysis with known positive controls (HepG2 or SMMC-7721 cells) and negative controls (cells with confirmed ZNF253 knockdown). Western blot analysis should demonstrate the expected molecular weight band (58-65 kDa) without significant additional bands. For more stringent validation, implement immunoprecipitation followed by mass spectrometry to confirm antibody specificity. Recent advances in antibody design allow for computational approaches to enhance specificity profiles, particularly for distinguishing between chemically similar epitopes . Consider using orthogonal detection methods, such as expressing tagged versions of ZNF253 and detecting with tag-specific antibodies to confirm results obtained with ZNF253-specific antibodies. Additionally, peptide competition assays using the immunizing peptide can confirm binding specificity. For critical applications, compare results using multiple antibodies targeting different epitopes within ZNF253 to increase confidence in specificity.

What strategies can improve immunoprecipitation efficiency when studying ZNF253 protein interactions?

Optimizing immunoprecipitation (IP) efficiency for ZNF253 requires addressing several experimental variables. Begin with proper cell lysis conditions, using buffers containing 0.1-1% NP-40 or Triton X-100 to maintain protein interactions while ensuring effective extraction. Pre-clearing lysates with matched IgG (rabbit IgG for rabbit polyclonal ZNF253 antibodies) is essential to reduce non-specific binding . For the IP reaction, use 2-5 μg of antibody per 500 μg of total protein, and incubate overnight at 4°C with gentle rotation to maximize antigen-antibody interactions. Consider crosslinking the antibody to beads (protein A/G) to prevent antibody co-elution, which can interfere with subsequent analyses. For studying transient or weak interactions, incorporate crosslinking agents like formaldehyde or DSP before cell lysis. To enhance detection of binding partners, consider using label-free quantitative proteomics to identify proteins enriched in ZNF253 immunoprecipitates compared to IgG controls. Validate key interactions through reciprocal IP experiments and co-localization studies using immunofluorescence microscopy, following protocols similar to those documented for related zinc finger proteins .

How do I interpret conflicting ZNF253 expression data across different cancer cell lines?

Interpreting conflicting ZNF253 expression data across cancer cell lines requires systematic analysis of multiple factors. First, validate detection methods by comparing protein expression (Western blot) with mRNA expression (qRT-PCR) to determine if discrepancies arise from post-transcriptional regulation. Consider cell line authenticity and passage number, as genetic drift can affect gene expression profiles. Analyze cell culture conditions, as serum components, confluency, and oxygen levels can significantly alter transcription factor expression. Evaluate the subcellular localization of ZNF253 using fractionation techniques or immunofluorescence microscopy, as nuclear versus cytoplasmic distribution may vary between cell lines, affecting detection . Compare your findings with publicly available databases like TCGA, similar to approaches used for related zinc finger proteins where expression correlations with target genes have been established . Investigate potential regulators of ZNF253 expression in different cellular contexts, including microRNAs, promoter methylation, and upstream signaling pathways. Finally, consider functional redundancy with other zinc finger family members, which may compensate for or mask ZNF253 activity in certain cell types.

What controls should be included when using ZNF253 antibodies for immunofluorescence studies?

For rigorous immunofluorescence studies with ZNF253 antibodies, implement a comprehensive set of controls. Primary controls should include known ZNF253-positive cell lines such as HepG2 or SMMC-7721 , alongside negative controls using cells where ZNF253 has been knocked down via siRNA or shRNA. Secondary antibody-only controls are essential to assess non-specific binding, applying the secondary antibody without the primary ZNF253 antibody . Include an isotype control using an irrelevant primary antibody of the same isotype (rabbit IgG for rabbit polyclonal ZNF253 antibodies) to evaluate non-specific binding . For subcellular localization studies, incorporate counterstaining with DAPI for nuclear visualization and additional markers for specific cellular compartments. To confirm antibody specificity, perform peptide competition assays by pre-incubating the antibody with the immunizing peptide. For signal validation, compare the staining pattern with tagged ZNF253 expression constructs detected via the tag. Document microscope settings consistently across all samples and controls, including exposure times, gain settings, and post-acquisition processing parameters to ensure objective data interpretation.

How can I optimize Western blotting protocols specifically for ZNF253 detection?

To optimize Western blotting for ZNF253 detection, adapt several key parameters of standard protocols. Begin with sample preparation: use RIPA buffer supplemented with protease inhibitors for cell lysis, and heat samples at 95°C for 5 minutes in Laemmli buffer containing DTT for complete protein denaturation. For gel electrophoresis, 10% SDS-PAGE gels provide optimal resolution in the 58-65 kDa range where ZNF253 is detected . During transfer, use PVDF membranes which typically offer better protein retention than nitrocellulose for transcription factors. Block membranes with 5% non-fat dry milk or BSA in TBST for 1 hour at room temperature. For primary antibody incubation, start with a 1:500 dilution of ZNF253 antibody in blocking buffer overnight at 4°C , then optimize based on signal-to-noise ratio. Use HRP-conjugated secondary antibodies at 1:5000 dilution for 1 hour at room temperature. For enhanced sensitivity, consider using signal amplification systems or fluorescent secondary antibodies with infrared detection. Include positive controls (HepG2 or SMMC-7721 lysates) and molecular weight markers to confirm the expected 58-65 kDa band size. For troubleshooting, extend exposure times for weak signals or implement gradient dilution series of both primary and secondary antibodies to determine optimal concentrations.

What are the best experimental approaches to confirm ZNF253 antibody specificity?

Confirming ZNF253 antibody specificity requires multiple complementary approaches. Begin with genetic validation by testing the antibody in ZNF253 knockdown/knockout systems using siRNA, shRNA, or CRISPR-Cas9 technologies, which should show reduced or absent signal compared to control cells . Perform peptide competition assays by pre-incubating the antibody with excess immunizing peptide, which should abolish specific binding. For cross-reactivity assessment, test the antibody against recombinant proteins of closely related zinc finger family members, especially those with high sequence homology. Implement immunoprecipitation followed by mass spectrometry to identify all proteins captured by the antibody. Compare results across multiple ZNF253 antibodies targeting different epitopes; concordant results strengthen specificity confidence. For further validation, express tagged versions of ZNF253 in cellular systems and confirm co-localization of tag-specific and ZNF253-specific antibody signals. Advanced computational approaches to antibody design and validation can also help distinguish between similar epitopes . Document all validation experiments thoroughly, including positive and negative controls, to establish a robust profile of antibody specificity under various experimental conditions.

What sample preparation techniques are optimal for preserving ZNF253 epitope integrity?

Preserving ZNF253 epitope integrity during sample preparation requires careful consideration of multiple factors. For protein extraction, use gentle lysis buffers containing 0.1-1% non-ionic detergents (NP-40, Triton X-100) rather than stronger ionic detergents that may denature proteins. Include protease inhibitor cocktails to prevent epitope degradation and phosphatase inhibitors if studying phosphorylation status. Maintain samples at 4°C throughout processing to minimize proteolytic degradation. For fixed tissue samples, optimize fixation conditions with 4% paraformaldehyde for 10 minutes rather than longer periods or harsher fixatives like glutaraldehyde, which can mask epitopes . For immunohistochemistry applications, consider antigen retrieval methods using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0) with controlled heating to recover epitopes while maintaining tissue morphology. When freezing samples for long-term storage, use rapid freezing in liquid nitrogen followed by storage at -80°C, and avoid repeated freeze-thaw cycles. For Western blotting, limit sample heating time and temperature during denaturation (95°C for 5 minutes maximum), as prolonged heating can destroy epitopes. When using FFPE tissue sections, prepare 4 μm thick sections for optimal antibody penetration and epitope accessibility .

How can ZNF253 antibodies be utilized in cancer research, particularly for breast cancer studies?

ZNF253 antibodies offer valuable tools for investigating potential roles in cancer biology, particularly in breast cancer research. For triple negative breast cancer (TNBC) studies, implement immunohistochemistry protocols similar to those documented for related zinc finger proteins in clinical samples to assess correlation with clinical parameters and patient outcomes . Analyze ZNF253 expression patterns across breast cancer subtypes using tissue microarrays and validated antibodies at optimized dilutions. To investigate functional roles, combine ZNF253 knockdown or overexpression in breast cancer cell lines with phenotypic assays measuring proliferation, migration, and invasion. ChIP-seq experiments using ZNF253 antibodies can identify cancer-relevant target genes and pathways regulated by this transcription factor. Co-immunoprecipitation studies may reveal interactions with known oncogenic or tumor suppressor pathways, similar to documented interactions between YAP and other zinc finger proteins in TNBC . Analyze correlations between ZNF253 expression and established breast cancer biomarkers or therapeutic targets using bioinformatic approaches with TCGA datasets, following methods used for related zinc finger proteins . For translational relevance, examine ZNF253 expression changes in response to standard chemotherapeutic agents or targeted therapies to identify potential roles in treatment response mechanisms.

What is the significance of ZNF253's C2H2-type zinc fingers and KRAB domain in experimental design?

The presence of eleven C2H2-type zinc fingers and a KRAB domain in ZNF253 has significant implications for experimental design . C2H2-type zinc fingers typically mediate sequence-specific DNA binding, suggesting experiments should focus on identifying genomic targets through ChIP-seq or similar approaches. Design DNA-binding experiments with attention to zinc-dependent folding by including zinc in buffers (typically 10-50 μM ZnCl₂) to maintain finger structure. The KRAB domain commonly functions as a transcriptional repressor by recruiting co-repressor complexes , prompting investigation of interactions with known KRAB-associated proteins like KAP1/TRIM28 through co-immunoprecipitation studies. For functional studies, create domain deletion constructs separating the KRAB domain from zinc finger regions to dissect their individual contributions to ZNF253 activity. When analyzing protein-protein interactions, consider the KRAB domain as a potential protein interaction interface. For evolutionary studies, compare ZNF253 sequence conservation across species, focusing separately on KRAB domains and zinc finger regions, which often evolve at different rates. When developing therapeutic approaches targeting ZNF253, evaluate the accessibility of these domains to small molecules or peptides, as zinc finger domains offer potential for selective targeting.

How can I investigate potential post-translational modifications of ZNF253 using available antibodies?

Investigating post-translational modifications (PTMs) of ZNF253 requires specialized experimental approaches leveraging available antibodies. Begin with immunoprecipitation using validated ZNF253 antibodies followed by mass spectrometry analysis to identify PTM sites. The observed molecular weight range of 58-65 kDa versus the calculated 58 kDa suggests potential modifications . For phosphorylation studies, immunoprecipitate ZNF253 and probe with phospho-specific antibodies (anti-phosphoserine, anti-phosphothreonine, anti-phosphotyrosine) or perform phosphatase treatment to observe mobility shifts on SDS-PAGE. To investigate SUMOylation or ubiquitination, perform immunoprecipitation under denaturing conditions to preserve these modifications, followed by Western blotting with anti-SUMO or anti-ubiquitin antibodies. For acetylation studies, treat cells with histone deacetylase inhibitors before immunoprecipitation to enhance detection of acetylated forms. Develop site-specific antibodies for identified PTM sites to monitor modification status under various cellular conditions. Perform comparative analysis across different cell types, stress conditions, or cell cycle stages to identify regulatory patterns of ZNF253 modifications. For functional implications, create point mutations at identified PTM sites and assess effects on ZNF253 localization, stability, and transcriptional activity through reporter gene assays.

What strategies should be employed when studying ZNF253 in primary human tissues versus cell lines?

Studying ZNF253 in primary human tissues presents distinct challenges compared to cell line models, requiring tailored strategies. For tissue-based studies, optimize fixation protocols to preserve epitope accessibility—4% paraformaldehyde for 10 minutes has been successfully used for related zinc finger proteins . When working with limited primary material, implement signal amplification techniques such as tyramide signal amplification for immunohistochemistry or proximity ligation assays to detect protein-protein interactions with greater sensitivity. For FFPE tissues, standardize section thickness to 4 μm for consistent antibody penetration . Consider laser capture microdissection to isolate specific cell populations for subsequent protein or RNA analysis of ZNF253. For comparative studies, develop a comprehensive tissue panel to establish baseline expression patterns across different tissue types. When analyzing clinical samples, correlate ZNF253 expression with patient characteristics and outcomes, following approaches used for other zinc finger proteins in cancer research . For functional validation in primary contexts, consider ex vivo culture systems or patient-derived xenografts where genetic manipulation can be performed. Address tissue heterogeneity by incorporating multiplexed immunofluorescence approaches to simultaneously detect ZNF253 alongside cell-type-specific markers, allowing for cell type-specific expression analysis within complex tissues.

What are the essential positive and negative controls for ZNF253 antibody validation experiments?

For comprehensive ZNF253 antibody validation, implement a structured control system. Essential positive controls include lysates from HepG2 or SMMC-7721 cells, which have documented ZNF253 expression . For quantitative benchmarking, include recombinant ZNF253 protein at known concentrations to establish detection limits and linear range. Negative controls should feature lysates from cells with confirmed ZNF253 knockdown via siRNA or shRNA technologies , alongside isotype-matched control antibodies (rabbit IgG for rabbit polyclonal ZNF253 antibodies) to assess non-specific binding . For immunostaining applications, include secondary antibody-only controls to evaluate background signal . Peptide competition controls, where the antibody is pre-incubated with the immunizing peptide, should eliminate specific binding while non-specific interactions remain. For cross-reactivity assessment, test the antibody against closely related zinc finger proteins, particularly those with high sequence homology in the antibody's target epitope region. Include lysates from multiple tissue or cell types with varying expected expression levels to establish detection thresholds across biological contexts. Document all validation controls with appropriate technical replicates and quantitative analysis to establish robust validation metrics for each experimental application.

How should I interpret and troubleshoot unexpected band patterns in ZNF253 Western blots?

When encountering unexpected band patterns in ZNF253 Western blots, implement a systematic troubleshooting approach. If additional bands appear, determine if they represent isoforms, post-translational modifications, or degradation products by comparing patterns across sample types and preparation methods. The expected molecular weight for ZNF253 is 58-65 kDa ; bands significantly deviating from this range warrant investigation. For bands larger than expected, consider potential post-translational modifications such as SUMOylation or ubiquitination, and verify through immunoprecipitation followed by modification-specific antibody detection. For smaller bands, examine potential proteolytic cleavage by adding increasing concentrations of protease inhibitors during sample preparation. To distinguish between specific and non-specific bands, perform peptide competition assays and siRNA knockdown experiments, where specific bands should show reduced intensity. For persistent non-specific bands, optimize antibody concentration (try higher dilutions from 1:500 to 1:1000) , extend blocking time, or test alternative blocking reagents. Consider sample preparation variables including lysis buffer composition, protein denaturation conditions, and gel percentage. For definitive identification of ambiguous bands, excise the gel region containing the band of interest and perform mass spectrometry analysis to confirm protein identity.

What techniques can confirm the specificity of ZNF253 antibodies for detecting endogenous versus overexpressed protein?

Confirming antibody specificity for endogenous versus overexpressed ZNF253 requires multiple complementary approaches. Begin with side-by-side Western blot analysis of endogenous expression in validated positive cell lines (HepG2, SMMC-7721) alongside cells transfected with ZNF253 expression constructs at varying levels. Implement titration experiments with increasing amounts of transfected ZNF253 to assess detection linearity and potential artifacts at high expression levels. For definitive validation, perform parallel knockdown experiments using siRNA or shRNA targeting endogenous ZNF253 , which should reduce or eliminate the specific band while not affecting bands from overexpression constructs containing silent mutations at the siRNA target site. Use epitope-tagged overexpression constructs to perform dual detection with both ZNF253 antibodies and tag-specific antibodies, confirming signal overlap. For immunofluorescence applications, compare subcellular localization patterns between endogenous and overexpressed protein to identify potential artifacts of overexpression. Consider pulse-chase experiments with metabolic labeling to distinguish newly synthesized overexpressed protein from stable endogenous protein pools. For quantitative assessment, develop standard curves using purified recombinant protein to determine absolute detection limits and compare sensitivity for detecting endogenous versus overexpressed ZNF253 across multiple experimental techniques.

How can I verify if my experimental results with ZNF253 antibodies align with known biological functions of zinc finger proteins?

To verify alignment between experimental results and known zinc finger protein biology, implement a multilayered validation approach. First, assess ZNF253's subcellular localization through immunofluorescence studies, confirming predominant nuclear localization consistent with transcription factor function . Examine co-localization with chromatin markers during different cell cycle phases to verify DNA-binding characteristics typical of C2H2 zinc finger proteins. For functional validation, perform ChIP experiments followed by sequencing or qPCR of candidate targets, confirming binding to DNA sequences containing motifs consistent with C2H2 zinc finger recognition patterns. Analyze the effect of zinc chelation on ZNF253 function, as proper zinc finger folding is zinc-dependent. Investigate protein interactions through co-immunoprecipitation studies, focusing on known interactors of KRAB domain-containing proteins such as KAP1/TRIM28 . Compare expression patterns across tissue types with those of related zinc finger proteins to identify conserved regulatory networks. For pathway integration, examine interactions with established transcriptional machinery components. Evaluate responses to stimuli known to modulate other zinc finger protein activities, such as cellular stress, growth factors, or differentiation signals. Cross-reference experimental findings with bioinformatic predictions of ZNF253 functions based on domain structure and evolutionary conservation to establish coherence with the broader zinc finger protein family biology.