IKZF1 Recombinant Monoclonal Antibody

What is IKZF1 and why is it significant in research?

IKZF1, also known as DNA-binding protein Ikaros, belongs to the Ikaros C2H2-type zinc-finger protein family. It functions as a transcription regulator of hematopoietic cell differentiation. The N-terminal zinc-fingers 2 and 3 are required for DNA binding and targeting to pericentromeric heterochromatin, while the C-terminal zinc-finger domain facilitates dimerization. IKZF1 is abundantly expressed in thymus, spleen, peripheral blood leukocytes, and lymph nodes, where it plays a crucial role in lymphocyte development . Its significance in research stems from its involvement in lymphoid malignancies and its role as a master regulator of lymphocyte differentiation, making it an important target for immunological and cancer research.

Which cell lines and tissues are optimal for IKZF1 antibody validation?

For antibody validation, several cell lines and tissues show robust IKZF1 expression and serve as excellent positive controls:

These samples provide reliable sources for validating IKZF1 antibodies across different experimental applications . When establishing a new experimental system, testing these validated positive controls alongside your samples of interest helps ensure antibody functionality.

How should researchers optimize Western blot protocols for IKZF1 detection?

For optimal Western blot detection of IKZF1, researchers should consider the following methodology:

• Antibody dilution: The recommended dilution range for IKZF1 monoclonal antibody (66966-1-Ig) is 1:5000-1:50000 . This wide range reflects sample-dependent variability.

• Sample preparation: Protein extraction from nuclear fractions yields better results since IKZF1 is primarily localized in the nucleus.

• Denaturation conditions: Use standard SDS-PAGE denaturation (95°C for 5 minutes) in Laemmli buffer with β-mercaptoethanol.

• Expected molecular weight: Look for bands between 55-65 kDa, not precisely at the calculated 53 kDa .

• Controls: Include positive controls such as Jurkat or Raji cell lysates alongside experimental samples.

• Optimization: Each research system requires titration of the antibody to obtain optimal signal-to-noise ratio.

Researchers encountering detection issues should first verify protein extraction efficiency from nuclear fractions before adjusting antibody concentration or incubation conditions.

What considerations are important for immunofluorescence applications with IKZF1 antibodies?

For immunofluorescence/immunocytochemistry applications using fluorescently conjugated IKZF1 antibodies:

• Antibody selection: Consider using CoraLite® Plus 488-conjugated IKZF1 monoclonal antibody for direct detection without secondary antibodies .

• Dilution range: Start with 1:50-1:500 dilution and optimize based on signal intensity .

• Fixation method: Use 4% paraformaldehyde fixation followed by Triton X-100 permeabilization to access nuclear antigens.

• Positive control: Jurkat cells serve as reliable positive controls for IF/ICC applications .

• Subcellular localization: Expect nuclear staining pattern with possible concentration in pericentromeric heterochromatin regions.

• Fluorescence properties: When using CoraLite® Plus 488, set excitation/emission wavelengths to 493 nm/522 nm respectively .

Nuclear counterstaining with DAPI helps visualize IKZF1's nuclear localization pattern and confirms proper sample preparation and antibody functionality.

How can flow cytometry protocols be optimized for intracellular IKZF1 detection?

For intracellular IKZF1 detection by flow cytometry:

• Sample preparation: Use freshly isolated cells when possible, with immediate fixation and permeabilization.

• Antibody amount: Use approximately 0.40 μg of CoraLite® Plus 488-conjugated IKZF1 antibody per 10^6 cells in a 100 μl suspension .

• Fixation/permeabilization: For intracellular staining, fix cells with 4% paraformaldehyde followed by permeabilization using a methanol-based or saponin-based buffer system.

• Controls: Include:

  • Unstained cells for autofluorescence assessment

  • Isotype controls (Mouse IgG1 conjugated to the same fluorophore)

  • FMO (Fluorescence Minus One) controls

  • Positive control samples (human PBMCs)

• Gating strategy: First gate on lymphocytes based on FSC/SSC, then exclude doublets and dead cells before analyzing IKZF1 expression.

• Compensation: When multiplexing, proper compensation is essential due to the 493/522 nm excitation/emission profile of CoraLite® Plus 488.

This methodological approach ensures specific detection of intracellular IKZF1 with minimal background and false positives.

How should researchers interpret IKZF1 mutations in the context of lymphoid malignancies?

IKZF1 mutations are significant in lymphoid malignancies, with characteristic patterns researchers should consider during analysis:

• Mutation clustering: Somatic nonsynonymous mutations frequently cluster in N-terminal zinc fingers 2 and 3, which are essential for DNA-binding . This pattern suggests functional consequences for DNA binding rather than random mutational events.

• Mutation types: Analyze both point mutations and deletions, as IKZF1 can be altered through both mechanisms.

• Functional domains affected: When documenting mutations, map them to specific functional domains (DNA-binding zinc fingers vs. dimerization domains) as this affects functional outcomes .

• Isoform considerations: Consider which isoforms are affected by the mutations. Chicken Ikaros isoforms 1 and 2 closely resemble human isoforms (86% and 78% amino acid identity respectively) .

• Experimental validation: Functional validation of mutations through DNA-binding assays or reporter gene assays is essential to confirm pathogenicity.

When interpreting sequencing data, researchers should assess both the location and nature of IKZF1 mutations to accurately classify their potential impact on protein function and disease pathogenesis.

What are the optimal storage conditions for maintaining IKZF1 antibody activity?

To ensure long-term stability and functionality of IKZF1 antibodies:

• Storage temperature: Store antibodies at -20°C for long-term preservation. Both unconjugated and fluorescently-conjugated versions remain stable for one year after shipment under these conditions .

• Buffer composition:

  • The unconjugated antibody is provided in PBS with 0.02% sodium azide and 50% glycerol (pH 7.3) .

  • The CoraLite® Plus 488-conjugated antibody is in PBS with 50% Glycerol, 0.05% Proclin300, and 0.5% BSA (pH 7.3) .

• Aliquoting considerations:

  • For the unconjugated antibody, aliquoting is unnecessary for -20°C storage .

  • For the conjugated antibody, aliquoting is recommended to minimize freeze-thaw cycles, and vials should be protected from light exposure .

• Working solution preparation: Dilute only the amount needed for immediate experiments, and avoid repeated freeze-thaw cycles of the stock solution.

• Shipping considerations: Short-term shipping at ambient temperature is acceptable, but antibodies should be transferred to -20°C immediately upon receipt.

These storage protocols ensure maximum antibody performance and reproducibility across experiments.

How can IKZF1 recombinant antibodies be used to investigate protein-protein interactions?

To investigate IKZF1 protein-protein interactions:

• Co-immunoprecipitation (Co-IP): IKZF1 antibody 66966-1-Ig has been validated for Co-IP applications . The general protocol includes:

  • Preparation of nuclear extracts (where IKZF1 primarily localizes)

  • Pre-clearing with protein A/G beads

  • Incubation with IKZF1 antibody overnight at 4°C

  • Capture with fresh protein A/G beads

  • Stringent washing and elution

  • Analysis of co-precipitated proteins by Western blot or mass spectrometry

• Controls for Co-IP experiments:

  • Input sample (pre-immunoprecipitation lysate)

  • IgG control (mouse IgG1 isotype control)

  • Negative control (cell line with IKZF1 knockdown)

• Cross-linking considerations: For transient or weak interactions, consider using membrane-permeable crosslinkers prior to cell lysis.

• Alternative approaches:

  • Proximity ligation assay (PLA) using IKZF1 antibody paired with antibodies against suspected interaction partners

  • FRET analysis using fluorescently labeled antibodies

These methodological approaches enable investigation of both known and novel IKZF1 protein-protein interactions in different cellular contexts.

What are common troubleshooting strategies for IKZF1 antibody applications?

When encountering issues with IKZF1 antibody performance, consider these methodological solutions:

• Western blot troubleshooting:

  • No signal: Check positive controls (Jurkat, Raji cells) ; increase antibody concentration; extend incubation time

  • Multiple bands: Optimize SDS-PAGE conditions; verify sample purity; consider isoform detection

  • Wrong molecular weight: Remember IKZF1 runs between 55-65 kDa, not exactly at 53 kDa

• Immunofluorescence troubleshooting:

  • High background: Increase blocking time/concentration; reduce primary antibody concentration

  • No signal: Ensure adequate permeabilization for nuclear antigen access; verify fixation protocol

  • Non-nuclear staining: Review fixation and permeabilization protocols; confirm antibody specificity

• Flow cytometry troubleshooting:

  • Poor separation: Optimize fixation/permeabilization for intracellular staining

  • Low signal: Increase antibody amount up to 0.40 μg per 10^6 cells

  • High background: Improve washing steps; adjust compensation settings

• Antibody validation approaches:

  • Genetic validation: Test antibody in IKZF1 knockout/knockdown systems

  • Peptide competition: Pre-incubate antibody with immunizing peptide

  • Cross-reactivity testing: Test across species (antibody works with human, mouse, pig, rabbit samples)

Systematic troubleshooting focusing on these application-specific approaches helps resolve most issues encountered with IKZF1 antibody applications.

How can researchers validate IKZF1 antibody specificity in their experimental systems?

To validate IKZF1 antibody specificity for a particular experimental system:

• Positive and negative control samples:

  • Positive controls: Use cell lines with known IKZF1 expression (Raji, Jurkat, MOLT-4, Ramos, Daudi cells)

  • Negative controls: Use non-lymphoid cell lines or IKZF1 knockdown/knockout models

• Molecular validation methods:

  • Western blot: Confirm single band at expected molecular weight (55-65 kDa)

  • Mass spectrometry: Validate immunoprecipitated protein identity

  • Immunodepletion: Sequential immunoprecipitation should deplete IKZF1 signal

• Cross-reactivity assessment:

  • Test antibody across multiple species if working with non-human models

  • Verify reactivity with human, mouse, pig, and rabbit samples as documented

• Isoform detection verification:

  • Use recombinant IKZF1 isoforms as standards

  • Compare detection patterns in samples known to express different isoform distributions

• Functional validation:

  • Immunoprecipitation followed by functional assays (e.g., DNA-binding assay)

  • Chromatin immunoprecipitation for DNA-binding transcription factors

These validation steps ensure that experimental observations are truly attributable to IKZF1 and not to non-specific antibody interactions.

How can IKZF1 antibodies be applied to study hematological malignancies?

IKZF1 antibodies enable several methodological approaches for studying hematological malignancies:

• Expression profiling:

  • Western blot analysis of IKZF1 levels across patient samples and cell lines

  • Flow cytometry for single-cell analysis of IKZF1 expression in heterogeneous populations

  • Immunohistochemistry for tissue section analysis in lymphoid malignancies

• Mutation detection:

  • Immunoprecipitation followed by mass spectrometry to identify mutant IKZF1 proteins

  • Western blot to detect truncated variants or altered molecular weight species

  • Combining with genomic data to correlate mutations with protein expression

• Disease model applications:

  • Monitor IKZF1 in virus-induced lymphoma models like Marek's Disease Virus (MDV)

  • Assess IKZF1 status in B-cell malignancy models, particularly acute lymphoblastic leukemia (ALL)

  • Track treatment response through IKZF1 expression/localization changes

• Clinical correlation studies:

  • Stratify patient samples based on IKZF1 status

  • Correlate IKZF1 alterations with treatment response and prognosis

  • Identify subgroups with distinct molecular pathology

Research indicates that somatic mutations in IKZF1 are frequent in certain lymphomas, with 9 of 22 tumors in one study containing mutations that cluster in DNA-binding domains , making antibody-based detection methods valuable for categorizing disease subtypes.

What are the considerations for using IKZF1 antibodies in chromatin immunoprecipitation (ChIP) experiments?

For successful chromatin immunoprecipitation using IKZF1 antibodies:

• Experimental design considerations:

  • Target selection: IKZF1 binds gamma-satellite DNA and regulates lymphocyte development genes

  • Crosslinking optimization: Due to IKZF1's role in chromatin remodeling complexes, optimize formaldehyde crosslinking time (typically 10-15 minutes)

  • Sonication parameters: Aim for 200-500bp fragments for optimal resolution

• Antibody selection:

  • Choose antibodies targeting N-terminal regions to avoid interference with DNA binding

  • Validate antibody ChIP efficiency with known IKZF1 binding sites

  • Consider using multiple antibodies targeting different epitopes for validation

• Controls:

  • Input chromatin (pre-immunoprecipitation)

  • IgG control (mouse IgG1 isotype)

  • Positive control loci (well-established IKZF1 binding sites)

  • Negative control loci (regions without IKZF1 binding)

• Data analysis approaches:

  • Compare enrichment patterns across different cell types/conditions

  • Correlate with transcriptomic data to establish functional relationships

  • Integrate with other transcription factor ChIP data to identify cooperative binding

This methodological approach allows researchers to map IKZF1 binding sites genome-wide and understand its role in transcriptional regulation during normal development and in disease states.