ZIM2 Antibody

What is ZIM2 and what is its significance in genomic research?

ZIM2 (Zinc finger imprinted 2, also known as ZNF656) is a transcriptional regulator of particular interest in genomic imprinting research. In humans, ZIM2 shares a set of 5' exons and a common promoter with PEG3 (Paternally Expressed Gene 3), and both genes are paternally expressed . This makes ZIM2 significant for studies investigating genomic imprinting mechanisms, gene regulation, and evolutionary biology. The protein contains zinc finger domains that enable DNA binding and transcriptional regulation functions, particularly in the negative regulation of transcription by RNA polymerase II . ZIM2's nuclear localization and role in transcriptional regulation make it relevant for research into gene expression control mechanisms.

How does human ZIM2 differ structurally from its counterparts in other mammals?

Human ZIM2 exhibits unique structural characteristics compared to its orthologs in other mammals. In humans, ZIM2 and PEG3 are distinct genes that share 5' exons and a common promoter, with alternative splicing events connecting these shared exons either with the 4 exons unique to ZIM2 or with the 2 exons unique to PEG3 . This arrangement is species-specific, as mouse and cow ZIM2 and PEG3 genes do not share exons in common . Additionally, the imprinting status of ZIM2 is not conserved across mammalian species . Human ZIM2 also has multiple 5' alternatively spliced transcripts that encode the same protein . These structural differences make human ZIM2 an interesting subject for evolutionary and comparative genomics studies.

What are the primary applications for ZIM2 antibodies in research settings?

ZIM2 antibodies are versatile research tools applicable across multiple experimental techniques. Based on the available ZIM2 antibody clones, researchers can utilize these antibodies in various applications:

These applications enable researchers to investigate ZIM2 expression patterns, subcellular localization, protein interactions, and functional roles in various cellular contexts .

What are the molecular characteristics of the ZIM2 protein relevant to antibody selection?

ZIM2 has several key molecular characteristics that researchers should consider when selecting antibodies:

• Theoretical molecular weight: 61 kDa (61.16 kDa specifically)

• Primary cellular location: Nucleus

• Protein function: Transcriptional regulation, particularly negative regulation of transcription by RNA polymerase II

• Human specificity: Most commercially available antibodies are specific to human ZIM2

When selecting antibodies, researchers should consider these characteristics and verify that the antibody epitope aligns with their experimental needs. The molecular weight is particularly important for Western blot applications to confirm detection of the correct protein band. The nuclear localization is crucial for immunostaining experiments to validate proper subcellular localization patterns.

How should researchers validate ZIM2 antibody specificity for experimental applications?

Validating ZIM2 antibody specificity is critical for ensuring reliable experimental results. A comprehensive validation approach should include:

• Positive and negative controls: Use cell lines or tissues known to express or not express ZIM2. For example, transfected cells (as shown in the Western blot data with HEK293T cells transfected with pCMV6-ENTRY ZIM2) serve as excellent positive controls, while the empty vector transfection provides a suitable negative control .

• Knockdown/knockout validation: Perform siRNA knockdown or CRISPR-Cas9 knockout of ZIM2 to confirm antibody specificity. The disappearance or reduction of signal following knockdown/knockout provides strong evidence of specificity.

• Cross-reactivity testing: Test the antibody against related zinc finger proteins, particularly PEG3, given their shared exons and promoter in humans .

• Epitope verification: Confirm that the antibody recognizes the expected epitope. For instance, antibodies targeting the human recombinant protein fragment corresponding to amino acids 1-150 and 428-527 (as with OTI7G1) should be validated within these regions .

• Multiple detection methods: Validate using at least two different methods (e.g., Western blot and immunofluorescence) to confirm consistent protein detection patterns.

What are the optimal protocols for detecting ZIM2 in Western blot experiments?

For optimal Western blot detection of ZIM2, researchers should follow these recommended protocols:

• Sample preparation:

  • Use nuclear extracts rather than whole cell lysates for enriched detection

  • Include protease inhibitors to prevent protein degradation

  • Prepare samples in reducing conditions with SDS

• Gel electrophoresis and transfer:

  • Use 8-10% SDS-PAGE gels to properly resolve the 61 kDa ZIM2 protein

  • Transfer to PVDF membranes at 100V for 60-90 minutes

• Blocking and antibody incubation:

  • Block with 5% non-fat dry milk in TBST for 1 hour at room temperature

  • Dilute primary ZIM2 antibody 1:2000 (for OTI7G1) in blocking buffer

  • Incubate overnight at 4°C with gentle rocking

  • Wash 3-5 times with TBST

  • Incubate with appropriate HRP-conjugated secondary antibody (anti-mouse IgG for most ZIM2 antibodies) at 1:5000 for 1 hour at room temperature

• Detection:

  • Use enhanced chemiluminescence (ECL) for detection

  • Expect a band at approximately 61 kDa, though post-translational modifications may alter the observed molecular weight

• Controls:

  • Include positive control (e.g., cells transfected with ZIM2 expression construct)

  • Include negative control (e.g., cells transfected with empty vector)

How can researchers optimize immunofluorescence protocols for ZIM2 detection?

Optimizing immunofluorescence for ZIM2 requires careful attention to fixation, permeabilization, and antibody incubation conditions:

• Cell preparation and fixation:

  • Culture cells on glass coverslips or chamber slides

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

  • For ZIM2, which is nuclear, ensure thorough permeabilization with 0.25% Triton X-100 for 10 minutes

• Blocking and antibody incubation:

  • Block with 5% normal serum (matching the host of the secondary antibody) in PBS containing 0.1% Triton X-100 for 1 hour

  • Dilute primary ZIM2 antibody 1:100 in blocking solution

  • Incubate overnight at 4°C in a humidified chamber

  • Wash 3-5 times with PBS

  • Incubate with fluorophore-conjugated secondary antibody at 1:500 for 1 hour at room temperature in the dark

• Nuclear counterstaining and mounting:

  • Counterstain nuclei with DAPI (1 μg/ml) for 5 minutes

  • Mount with anti-fade mounting medium

• Controls and validation:

  • Include transfected controls (as shown in data with COS7 cells transiently transfected with pCMV6-ENTRY ZIM2)

  • Perform parallel staining omitting primary antibody

  • Co-stain with other nuclear markers to confirm localization

• Imaging considerations:

  • ZIM2 should display nuclear localization

  • Use confocal microscopy for optimal resolution of nuclear structures

  • Capture Z-stacks to fully visualize nuclear distribution patterns

What are the key considerations for studying ZIM2 in genomic imprinting research?

Studying ZIM2 in genomic imprinting research requires specific methodological approaches due to its unique imprinting status and relationship with PEG3:

• Allele-specific expression analysis:

  • Design assays that can distinguish maternal versus paternal allele expression

  • Use single nucleotide polymorphisms (SNPs) within ZIM2 coding regions to differentiate alleles

  • Employ allele-specific RT-PCR or RNA sequencing to quantify expression from each parental allele

• Tissue specificity considerations:

  • Account for potential tissue-specific imprinting patterns

  • Include multiple tissue types in imprinting studies, as imprinting status can vary across tissues

• Species-specific experimental design:

  • Remember that ZIM2 imprinting status is not conserved across mammals

  • Design species-appropriate experiments considering the structural differences between human ZIM2 (which shares exons with PEG3) and other mammalian orthologs (which do not)

• Methylation analysis:

  • Analyze the methylation status of the shared promoter region between ZIM2 and PEG3

  • Use bisulfite sequencing or methylation-specific PCR to assess CpG island methylation patterns

  • Correlate methylation patterns with allele-specific expression data

• Alternative splicing analysis:

  • Design experiments to detect the alternative splicing events connecting shared exons with ZIM2-specific or PEG3-specific exons

  • Use RNA-seq or exon-specific RT-PCR to quantify different splice variants

How can researchers design experiments to investigate ZIM2's role in transcriptional regulation?

To investigate ZIM2's role in transcriptional regulation, researchers should consider these experimental approaches:

• Chromatin immunoprecipitation (ChIP) assays:

  • Use validated ZIM2 antibodies to perform ChIP followed by sequencing (ChIP-seq) or qPCR

  • Optimize fixation conditions for nuclear proteins (1% formaldehyde for 10 minutes)

  • Focus analysis on genes involved in RNA polymerase II-mediated transcription, given ZIM2's role in this process

• Transcriptional reporter assays:

  • Create luciferase reporter constructs with promoters of suspected ZIM2 target genes

  • Perform co-transfection experiments with ZIM2 expression vectors and reporter constructs

  • Measure luciferase activity to quantify ZIM2's impact on transcriptional activity

• CRISPR-based approaches:

  • Generate ZIM2 knockout cell lines using CRISPR-Cas9

  • Perform RNA-seq to identify differentially expressed genes

  • Conduct rescue experiments with wild-type ZIM2 and mutant constructs lacking zinc finger domains

• Protein-protein interaction studies:

  • Use co-immunoprecipitation with ZIM2 antibodies to identify interacting transcriptional complexes

  • Consider the 1F10 clone which has been validated for immunoprecipitation applications

  • Perform proximity ligation assays to confirm interactions in intact cells

• Domain-specific functional analysis:

  • Create constructs with mutations in specific zinc finger domains

  • Test the impact of these mutations on DNA binding and transcriptional regulation

  • Map the functional domains required for transcriptional repression activities

How can researchers troubleshoot non-specific binding or high background with ZIM2 antibodies?

When encountering non-specific binding or high background with ZIM2 antibodies, consider these troubleshooting approaches:

• Western blot troubleshooting:

  • Increase blocking time or blocking agent concentration (try 5% BSA instead of milk)

  • Optimize antibody dilution (try 1:5000 instead of 1:2000)

  • Increase washing duration and frequency (5 washes of 5 minutes each)

  • Use freshly prepared buffers and reagents

  • Try alternative membrane types (PVDF vs. nitrocellulose)

• Immunofluorescence troubleshooting:

  • Optimize fixation conditions (try methanol fixation as an alternative)

  • Increase blocking time or serum concentration

  • Try different detergents for permeabilization (0.1% Saponin instead of Triton X-100)

  • Reduce primary antibody concentration or incubation time

  • Use centrifugation to clarify antibody solutions before use

• Flow cytometry troubleshooting:

  • Optimize fixation and permeabilization for nuclear proteins

  • Use appropriate isotype controls at the same concentration as the primary antibody

  • Titrate antibody concentration (usually starting at 1:100)

  • Analyze controls without primary antibody to assess background

  • Use proper compensation controls if performing multicolor analysis

• General considerations:

  • Test multiple antibody clones if available (OTI7G1, OTI5C7, etc.)

  • Validate antibody specificity using positive and negative controls

  • Consider pre-absorbing the antibody with recombinant protein to reduce non-specific binding

What are the considerations for comparing results across different ZIM2 antibody clones?

When comparing results obtained with different ZIM2 antibody clones, researchers should consider several important factors:

How should researchers optimize ZIM2 detection in specific tissue samples?

Optimizing ZIM2 detection in tissue samples requires consideration of tissue-specific factors:

• Tissue fixation optimization:

  • For formalin-fixed paraffin-embedded (FFPE) tissues, optimize antigen retrieval:

    • Test both heat-induced epitope retrieval (HIER) with citrate buffer (pH 6.0) and Tris-EDTA buffer (pH 9.0)

    • Try different retrieval times (10-30 minutes)

  • For frozen tissues, optimize fixation time with 4% PFA (5-15 minutes)

• Antibody concentration and incubation conditions:

  • Start with recommended dilution (1:150 for IHC with OTI7G1)

  • Perform antibody titration experiments to determine optimal concentration for each tissue type

  • Consider extended incubation times (overnight at 4°C) for improved sensitivity

• Signal detection methods:

  • Compare chromogenic (DAB) versus fluorescent detection systems

  • Use tyramide signal amplification for low-abundance targets

  • Consider multiplex IHC to co-localize with known nuclear markers

• Tissue-specific blocking optimization:

  • Use tissue-specific blocking agents (e.g., add avidin/biotin blocking for tissues with high endogenous biotin)

  • Include blocking steps for endogenous enzymes (peroxidase, phosphatase)

  • Use species-specific blocking serum matching the host of secondary antibody

• Validation in relevant tissues:

  • Validate staining patterns in tissues known to express ZIM2

  • Compare with existing literature or public database expression data

  • Consider the human lymph node tissue as a reference, which has been validated for ZIM2 antibody OTI7G1

How can researchers utilize ZIM2 antibodies in studying gene regulation networks?

ZIM2 antibodies can be powerful tools for investigating gene regulation networks through these methodological approaches:

• Integrated ChIP-seq and RNA-seq analysis:

  • Perform ChIP-seq with ZIM2 antibodies to identify genome-wide binding sites

  • Correlate binding data with RNA-seq from ZIM2 overexpression or knockdown experiments

  • Identify direct transcriptional targets versus secondary effects

  • Use bioinformatic approaches to identify DNA binding motifs and co-factors

• Multi-omics experimental design:

  • Combine ChIP-seq, RNA-seq, and proteomics approaches

  • Include ZIM2 antibody-based immunoprecipitation for protein complex identification

  • Map the complete network of ZIM2-regulated genes and interacting proteins

  • Analyze data using systems biology approaches to identify regulatory modules

• Single-cell approaches:

  • Apply ZIM2 antibodies in single-cell protein analysis using mass cytometry or imaging mass cytometry

  • Correlate with single-cell RNA-seq data to study cell-to-cell variability in ZIM2 function

  • Examine heterogeneity in ZIM2 expression and localization within tissues

• Temporal regulation studies:

  • Design time-course experiments using ZIM2 antibodies

  • Analyze dynamic changes in ZIM2 binding during cellular differentiation or response to stimuli

  • Correlate with temporal transcriptome changes to build dynamic regulatory networks

What considerations are important when analyzing ZIM2 in the context of its relationship with PEG3?

When analyzing ZIM2 in relation to PEG3, researchers should consider these methodological approaches:

• Distinguishing between ZIM2 and PEG3:

  • Design experiments that can differentiate between these genes despite their shared exons

  • Use antibodies targeting unique regions of each protein

  • Design PCR primers or RNA probes specific to unique exons

  • Verify specificity using controls expressing only ZIM2 or only PEG3

• Co-expression analysis:

  • Investigate whether ZIM2 and PEG3 are co-expressed in the same cells/tissues

  • Use dual immunofluorescence with antibodies specific to unique regions of each protein

  • Perform single-cell RNA-seq to examine co-expression at the cellular level

• Promoter regulation studies:

  • Analyze the shared promoter regulation mechanisms

  • Design reporter constructs with the shared promoter region

  • Perform ChIP assays to identify transcription factors binding to the shared promoter

  • Investigate epigenetic modifications of the shared promoter region

• Alternative splicing regulation:

  • Study the regulation of alternative splicing events that determine ZIM2 versus PEG3 expression

  • Identify splicing factors that regulate the choice between ZIM2 and PEG3 exons

  • Design minigene constructs to study splicing regulation in vitro

• Evolutionary analysis:

  • Compare human ZIM2/PEG3 organization with other species where they don't share exons

  • Investigate the evolutionary forces that led to the shared exon structure in humans

  • Examine functional consequences of the shared versus separate gene arrangements

What are the best practices for reporting ZIM2 antibody usage in scientific publications?

When reporting ZIM2 antibody usage in scientific publications, researchers should adhere to these best practices:

• Comprehensive antibody information:

  • Report complete antibody information including:

    • Clone number (e.g., OTI7G1, 1C2)

    • Host species and isotype (e.g., Mouse IgG1)

    • Supplier and catalog number

    • RRID (Research Resource Identifier) when available (e.g., AB_2619298)

  • Describe the target epitope if known (e.g., amino acids 1-150 and 428-527)

• Detailed methodology:

  • Provide complete experimental protocols including:

    • Sample preparation methods

    • Antibody dilutions used for each application

    • Incubation conditions (time, temperature)

    • Detection methods and imaging parameters

  • Include information about controls and validation experiments

• Validation documentation:

  • Report how antibody specificity was validated

  • Include positive and negative control data

  • Document any observed cross-reactivity

  • Reference previous publications that have validated the antibody

• Results interpretation guidelines:

  • Discuss potential limitations of the antibody-based detection

  • Address possible alternative interpretations of results

  • Acknowledge any discrepancies between antibody-based results and other methods

• Data availability:

  • Include unprocessed blot/gel images as supplementary material

  • Deposit raw microscopy data in appropriate repositories

  • Make detailed protocols available through protocol repositories