PRDM16 Antibody, FITC conjugated

What is PRDM16 and why is it an important research target?

PRDM16 (PR Domain Containing 16) is a zinc finger transcription factor containing an N-terminal PR domain. It plays critical roles in multiple biological processes, making it a significant target for research. PRDM16 has been shown to be essential for stem cell maintenance throughout the nervous and hematopoietic systems . Additionally, PRDM16 is involved in the pathogenesis of myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML), particularly in cases with the reciprocal translocation t(1;3)(p36;q21) . This protein has also been identified as a Smad3 binding protein that modulates the TGF-beta signaling pathway, which is important for the development of orofacial structures . Furthermore, PRDM16 controls a bidirectional cell fate switch between skeletal myoblasts and brown fat cells, highlighting its significance in cellular differentiation research .

How does the molecular structure of PRDM16 influence antibody selection and experimental design?

PRDM16's complex molecular structure significantly impacts antibody selection and experimental design decisions. The full-length human PRDM16 is a large protein with a calculated molecular weight of approximately 140 kDa, though it is typically observed at ~150-170 kDa on Western blots due to post-translational modifications . When designing experiments, researchers must consider:

• Domain-specific targeting: PRDM16 contains multiple functional domains, including the PR (PRDI-BF1 and RIZ homology) domain, zinc finger domains, and regions mediating protein-protein interactions. Antibodies targeting different epitopes may yield varying results based on protein conformation and interaction status .

• Isoform consideration: The truncated version of PRDM16 lacking the PR domain (observed in certain leukemias) may not be detected by antibodies targeting the N-terminal region . When studying disease models, researchers should select antibodies that recognize relevant regions of the protein.

• Species cross-reactivity: Available PRDM16 antibodies show different cross-reactivity profiles, with some recognizing only human PRDM16 while others detect human, mouse, and rat orthologs . This must be considered when designing experiments with animal models.

The amino acid region recognized by the antibody is also critical. For instance, the FITC-conjugated antibody described in search result targets amino acids 448-683, which affects what conformations and complexes of PRDM16 it can detect in experimental conditions.

What are the validated applications for PRDM16 antibodies, and how do they differ for FITC-conjugated versions?

PRDM16 antibodies have been validated across multiple applications, with conjugation status affecting optimal usage. Based on the search results, the following applications have been validated for various PRDM16 antibodies:

How should researchers optimize immunofluorescence protocols when using FITC-conjugated PRDM16 antibodies?

Optimizing immunofluorescence protocols with FITC-conjugated PRDM16 antibodies requires attention to several methodological considerations:

• Fixation method selection: The choice between paraformaldehyde, methanol, or acetone fixation can significantly impact epitope accessibility. For nuclear transcription factors like PRDM16, methanol fixation often provides better nuclear permeabilization and epitope preservation .

• Antigen retrieval optimization: For fixed tissue sections, heat-induced epitope retrieval may be necessary. Based on the information in search result , TE buffer at pH 9.0 is recommended for PRDM16 detection, though citrate buffer at pH 6.0 provides an alternative approach.

• Blocking and permeabilization: Use 5-10% normal serum from the same species as the secondary antibody (if any) with 0.1-0.3% Triton X-100 for permeabilization, as PRDM16 is primarily a nuclear protein .

• Antibody dilution optimization: Start with manufacturer recommendations (e.g., 1:50-1:500 for IHC applications) and perform a dilution series to determine optimal signal-to-noise ratio . FITC-conjugated antibodies may require higher concentrations than enzymes-conjugated counterparts due to lower sensitivity.

• Photobleaching prevention: FITC is particularly susceptible to photobleaching. Use anti-fade mounting media containing DABCO or similar agents, minimize exposure to light during processing, and consider using shorter exposure times with higher antibody concentrations during image acquisition .

• Autofluorescence reduction: Tissue autofluorescence can interfere with FITC signal detection. Treatment with Sudan Black B (0.1-0.3% in 70% ethanol) after antibody incubation can reduce autofluorescence, particularly in tissues with high lipofuscin content.

• Counterstaining considerations: When choosing nuclear counterstains, avoid propidium iodide as its emission spectrum overlaps with FITC. DAPI or Hoechst stains provide better spectral separation for co-visualization with FITC-labeled structures.

What are the critical considerations for using PRDM16 antibodies in Western blotting applications?

When employing PRDM16 antibodies for Western blotting, researchers should address several critical methodological considerations:

• Protein extraction optimization: PRDM16 is a large nuclear protein (observed at ~150-170 kDa), requiring efficient nuclear extraction protocols . Standard RIPA buffers may be insufficient; consider using specialized nuclear extraction buffers containing DNase to release DNA-bound transcription factors.

• Sample preparation: Due to its large size, complete denaturation is essential. Use strong reducing conditions (add fresh DTT or β-mercaptoethanol) and heat samples at 95°C for 5-10 minutes. For difficult samples, consider using 8M urea buffers to ensure complete denaturation.

• Gel selection: Use low percentage gels (6-8% acrylamide) or gradient gels to properly resolve the high molecular weight protein . Transfer efficiency for large proteins may require extended transfer times or specialized transfer systems.

• Blocking optimization: 5% non-fat dry milk in TBST is generally effective, though for phospho-specific antibodies, BSA-based blocking solutions are preferred to avoid phosphatases present in milk .

• Antibody dilution and incubation: For PRDM16 detection, optimal dilutions range from 1:500-1:3000 depending on the specific antibody . Overnight incubation at 4°C often yields better results than shorter incubations at room temperature for nuclear proteins.

• Expected band size validation: PRDM16 appears at approximately 150-170 kDa . Be aware that truncated forms (lacking the PR domain) may appear at lower molecular weights, particularly in leukemia samples. Multiple bands may indicate isoforms, post-translational modifications, or degradation products.

• Positive control selection: Jurkat cells, K-562 cells, and NCI-H1299 cells have been verified to express detectable levels of PRDM16 and can serve as positive controls . When using new cell lines, verify PRDM16 expression levels, as the protein has generally low expression in most tissues .

How can PRDM16 antibodies be utilized in investigating stem cell maintenance and differentiation?

PRDM16 antibodies serve as crucial tools for investigating stem cell biology due to the protein's significant role in stem cell maintenance across multiple tissues. Research has demonstrated that Prdm16 is preferentially expressed by stem cells throughout the nervous and hematopoietic systems and is required for their maintenance . When designing experiments to study these processes, researchers should consider several methodological approaches:

• Lineage tracing studies: FITC-conjugated PRDM16 antibodies can be used in flow cytometry to isolate PRDM16-expressing stem cell populations for subsequent in vitro or in vivo characterization . This approach allows for functional analysis of specific stem cell populations across different developmental stages or disease states.

• Co-localization analyses: Immunofluorescence with FITC-conjugated PRDM16 antibodies combined with other stem cell markers can reveal spatial relationships within stem cell niches. This technique is particularly valuable for studying the ventricular zone (VZ) of the central nervous system, where neural stem/progenitor cells reside .

• ChIP-seq applications: Chromatin immunoprecipitation followed by sequencing using PRDM16 antibodies can identify direct transcriptional targets. Previous research has demonstrated that Prdm16 directly binds promoters of specific genes like Hgf, but not others like Mt2 . These studies can be expanded to genome-wide analyses to comprehensively map PRDM16 binding sites in different stem cell populations.

• ROS level analysis in stem cells: PRDM16 has been implicated in controlling reactive oxygen species (ROS) levels in neural stem/progenitor cells . Combining PRDM16 immunostaining with ROS-sensitive dyes or reporters can provide insights into the mechanisms by which PRDM16 protects stem cells from oxidative stress.

• Brown adipocyte differentiation studies: Given PRDM16's role in controlling the cell fate switch between skeletal myoblasts and brown fat cells , antibodies can be used to track PRDM16 expression during differentiation protocols, identifying key regulatory points in the differentiation process.

When conducting these studies, researchers should be aware that PRDM16 expression is generally low in most tissues , necessitating sensitive detection methods and appropriate positive controls.

What approaches can be used to distinguish between full-length PRDM16 and its truncated isoforms in leukemia research?

Distinguishing between full-length PRDM16 and its truncated isoforms is crucial in leukemia research, as the truncated version lacking the PR domain (resulting from the t(1;3)(p36;q21) translocation) is implicated in the pathogenesis of myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) . Researchers can employ several strategic approaches:

• Epitope-specific antibody selection: Choose antibodies targeting different regions of PRDM16. For comprehensive analysis, use antibodies recognizing:

  • N-terminal region (including the PR domain) - will detect only full-length protein

  • C-terminal region - will detect both full-length and truncated isoforms

  • Middle regions (e.g., AA 448-683) - may detect both forms depending on exact truncation points

• Western blot analysis: The full-length PRDM16 appears at approximately 150-170 kDa, while the truncated isoform will appear at a lower molecular weight . Running these proteins in lower percentage gels (6-8%) will provide better resolution between these variants.

• Isoform-specific PCR: Design primers that specifically amplify either the full-length or truncated transcript to quantify relative expression levels of each isoform.

• Functional assessment through co-immunoprecipitation: The truncated and full-length forms interact with different protein partners. Immunoprecipitation with PRDM16 antibodies followed by detection of associated proteins can provide functional distinction between isoforms .

• Chromatin occupancy profiling: ChIP-seq using antibodies recognizing different PRDM16 domains can reveal differential chromatin occupancy patterns between the full-length and truncated forms, as they may regulate different sets of target genes .

• Transcriptional reporter assays: Using reporter constructs for known PRDM16 target genes can help distinguish the differential transcriptional regulatory activity of the full-length versus truncated protein.

When examining patient samples, researchers should always include appropriate controls such as cell lines with known expression of specific PRDM16 isoforms. K-562 cells, which are derived from a chronic myelogenous leukemia patient, can serve as a useful positive control for PRDM16 expression studies in leukemia research .

How can PRDM16 antibodies be employed in multiplex immunofluorescence assays to study protein interaction networks?

Multiplex immunofluorescence using FITC-conjugated PRDM16 antibodies enables comprehensive analysis of protein interaction networks and cellular contexts. This approach is particularly valuable for understanding PRDM16's diverse roles in different tissues and disease states. Here's a methodological framework for designing these complex experiments:

This multiplex approach allows researchers to simultaneously visualize PRDM16 with its interaction partners and within its cellular and tissue context, providing deeper insights into its biological functions and regulatory networks.

What are common challenges in detecting PRDM16 in experimental systems, and how can researchers overcome them?

Detecting PRDM16 presents several technical challenges that researchers should anticipate and address methodically:

• Low endogenous expression levels: PRDM16 has low expression in most cells and tissues , making detection difficult. To overcome this challenge:

  • Use sensitive detection systems such as polymer-based HRP amplification for IHC/WB

  • Employ tyramine signal amplification for fluorescence applications

  • Consider enrichment steps (e.g., nuclear fraction isolation) prior to analysis

  • Use cell lines with verified PRDM16 expression (Jurkat, K-562, NCI-H1299) as positive controls

• High molecular weight impeding efficient transfer: At ~150-170 kDa, PRDM16 transfers inefficiently in Western blotting . Optimization strategies include:

  • Extending transfer time (overnight transfer at low voltage)

  • Reducing methanol concentration in transfer buffer (5-10% instead of 20%)

  • Using specialized transfer systems designed for high molecular weight proteins

  • Implementing PVDF membranes with higher protein binding capacity than nitrocellulose

• Nuclear localization requiring proper permeabilization: As a transcription factor, PRDM16 localizes primarily to the nucleus. To ensure adequate access:

  • Use stronger permeabilization agents (0.5% Triton X-100 or 0.1% SDS)

  • Consider dual fixation methods (paraformaldehyde followed by methanol)

  • Optimize antigen retrieval using TE buffer at pH 9.0 as recommended

• Protein-protein interactions masking epitopes: PRDM16 functions in multi-protein complexes that may obscure antibody binding sites. Solutions include:

  • Testing multiple antibodies targeting different epitopes

  • Using denaturing conditions for applications where native conformation isn't required

  • Performing antigen retrieval to expose masked epitopes

• Differentiating specific from non-specific binding: Given the low expression levels, distinguishing true signal from background is critical. Validation approaches include:

  • Performing peptide competition assays to confirm specificity

  • Using multiple antibodies targeting different epitopes and comparing patterns

  • Including negative controls (tissues or cells known not to express PRDM16)

  • Validating with orthogonal methods (e.g., correlating protein detection with mRNA expression)

• Photobleaching of FITC conjugates: FITC is particularly susceptible to photobleaching. Mitigation strategies include:

  • Using anti-fade mounting media with DABCO or propyl gallate

  • Minimizing exposure during microscopy (lower excitation intensity, shorter exposure times)

  • Capturing FITC channels first in multichannel imaging sequences

  • Considering photobleaching correction during image analysis

By systematically addressing these challenges, researchers can improve detection sensitivity and specificity for PRDM16, enabling more reliable experimental outcomes.

How should researchers interpret discrepancies in PRDM16 detection across different experimental methods?

When researchers encounter discrepancies in PRDM16 detection across different experimental methods, systematic analysis and interpretation are required. These inconsistencies may reflect biological realities rather than technical failures. Here's a framework for addressing such discrepancies:

• Method-specific epitope accessibility variations: Different techniques expose different protein epitopes, potentially leading to inconsistent detection. For example:

  • Formalin fixation for IHC may mask epitopes that are accessible in Western blotting

  • Native protein folding in immunoprecipitation may hide epitopes that are exposed after denaturation

  • The epitope recognized by the FITC-conjugated antibody (AA 448-683) may have differential accessibility depending on protein conformation

  Interpretation approach: Map detected/undetected patterns to specific epitopes and structural domains of PRDM16 to identify if discrepancies correlate with particular protein regions.

• Isoform and post-translational modification differences: PRDM16 exists in multiple forms, including the full-length and truncated versions associated with leukemia .

  Interpretation approach: Compare molecular weights observed in Western blotting (expected ~150 kDa) with what would be predicted for specific isoforms or modified forms. Consider using phosphatase or glycosidase treatments to determine if post-translational modifications contribute to detection differences.

• Context-dependent protein complex formation: PRDM16 functions within different protein complexes that may mask antibody binding sites in a context-dependent manner.

  Interpretation approach: Use techniques like proximity ligation assays or co-immunoprecipitation to identify if PRDM16 detection correlates with specific protein-protein interactions.

• Subcellular localization variations: As a transcription factor, PRDM16 primarily localizes to the nucleus, but its detection may vary between nuclear and cytoplasmic compartments.

  Interpretation approach: Perform subcellular fractionation followed by Western blotting to determine if discrepancies relate to differential localization. Compare with immunofluorescence results showing spatial distribution.

• Sensitivity threshold differences: Different techniques have varying detection limits, particularly relevant for PRDM16 which has low expression in most tissues .

  Interpretation approach: Perform dilution series experiments with positive controls to establish detection limits for each method. Use quantitative approaches (qPCR, quantitative proteomics) to objectively assess expression levels.

• Correlation with functional outcomes: Ultimately, protein detection should correlate with expected biological functions.

  Interpretation approach: Assess if detected PRDM16 correlates with known downstream effects, such as target gene expression or cellular phenotypes (e.g., stem cell maintenance, adipocyte differentiation) .

When reporting discrepancies, researchers should provide comprehensive methodological details (antibody clone, epitope, dilution, detection system) and suggest biological interpretations based on the pattern of results, rather than simply dismissing certain results as "false negatives" or "false positives."

What are the best practices for validating PRDM16 antibody specificity in new experimental systems?

Rigorous validation of PRDM16 antibody specificity is essential when establishing new experimental systems, particularly given the complexity of this protein and its variable expression patterns. Researchers should implement a comprehensive validation strategy:

• Genetic validation approaches:

  • CRISPR/Cas9 knockout: Generate PRDM16 knockout cell lines as negative controls

  • siRNA/shRNA knockdown: Create transient knockdown models showing reduction in signal proportional to knockdown efficiency

  • Overexpression systems: Transfect cells with PRDM16 expression constructs to create positive controls with defined expression levels

• Epitope verification methods:

  • Peptide competition assays: Pre-incubate antibody with immunizing peptide to block specific binding

  • Multiple antibody comparison: Test antibodies targeting different PRDM16 epitopes (N-terminal, C-terminal, and middle regions)

  • Recombinant protein standards: Use purified recombinant PRDM16 as a standard for size verification

• Cross-species reactivity assessment:

  • Evaluate antibody performance across relevant species (human, mouse, rat) if cross-reactivity is claimed

  • Use species-specific positive controls (e.g., tissues or cells known to express PRDM16)

  • Sequence alignment analysis: Compare epitope sequences across species to predict cross-reactivity

• Multi-technique concordance:

  • Western blotting: Verify single band of expected molecular weight (~150-170 kDa)

  • Immunoprecipitation: Confirm pull-down of protein with expected size that can be recognized by other PRDM16 antibodies

  • Immunofluorescence/IHC: Verify expected subcellular localization (primarily nuclear) and tissue distribution pattern

  • Flow cytometry: Confirm specific staining in positive control cells versus negative controls

• Functional correlation validation:

  • ChIP-seq/ChIP-PCR: Verify binding to known PRDM16 target genes such as Hgf

  • Expression correlation: Compare protein detection with mRNA expression data from RT-qPCR or RNA-seq

  • Functional readouts: Correlate detection with known biological functions of PRDM16 in stem cell maintenance or adipocyte differentiation

• Protocol optimization for specific applications:

  • For FITC-conjugated antibodies: Determine optimal fixation and permeabilization conditions that preserve fluorescence while allowing epitope access

  • For tissues with high autofluorescence: Develop specific background reduction protocols

  • For low-expression contexts: Establish signal amplification approaches that maintain specificity

• Comprehensive documentation and reporting:

  • Document all validation experiments in detail, including positive and negative controls

  • Report antibody catalog number, lot number, and dilution factors used

  • Provide unedited images of blots and microscopy alongside analyzed data

By implementing these validation practices, researchers can establish confidence in their PRDM16 detection systems, enabling reliable interpretations of experimental results across different biological contexts.

How do PRDM16 antibodies compare to other detection methods for studying this protein's function?

When investigating PRDM16 function, antibody-based detection represents one of several methodological approaches, each with distinct advantages and limitations. Understanding these comparative differences enables researchers to select optimal approaches or design complementary multi-method strategies:

For optimal PRDM16 research, integration of multiple approaches provides the most comprehensive understanding:

• Initial characterization: Use antibodies for protein detection and localization , complemented by RNA analysis to identify expression patterns across tissues.

• Functional studies: Combine CRISPR-mediated modification with antibody detection to correlate protein presence/absence with phenotypic outcomes.

• Mechanistic investigation: Use ChIP-seq with PRDM16 antibodies to identify binding sites , then validate with reporter assays and genetic manipulation of target genes.

• Protein interaction networks: Begin with immunoprecipitation using PRDM16 antibodies followed by mass spectrometry, then validate specific interactions with co-immunoprecipitation and proximity ligation assays.

• Translational research: For clinical samples with limited material, use highly sensitive RNA detection methods for initial screening, followed by antibody-based validation in subset of samples.

This multi-method approach leverages the strengths of each technique while mitigating individual limitations, providing more robust and reproducible findings regarding PRDM16 function.

What emerging technologies might enhance or replace traditional antibody-based detection of PRDM16 in future research?

Several cutting-edge technologies are poised to transform PRDM16 research, either enhancing current antibody-based methods or providing alternative detection approaches:

• Proximity labeling proteomics (BioID, APEX):
  These methods involve expressing PRDM16 fused to a proximity-labeling enzyme that biotinylates nearby proteins, enabling identification of the entire PRDM16 interactome in living cells. This approach overcomes limitations of traditional co-immunoprecipitation by capturing even transient interactions and functions in native cellular environments. The resulting biotinylated proteins can be purified and identified by mass spectrometry, providing a comprehensive map of PRDM16's interaction network in different cellular contexts .

• Single-cell multi-omics integration:
  Integration of single-cell transcriptomics with single-cell proteomics (including antibody-based methods like CITE-seq) allows correlation of PRDM16 mRNA and protein levels at single-cell resolution. This technology is particularly valuable for studying heterogeneous stem cell populations where PRDM16 plays crucial regulatory roles . By simultaneously measuring PRDM16 expression, chromatin accessibility, and cell surface markers, researchers can identify cell populations where PRDM16 exerts its biological functions with unprecedented precision.

• Nanobody and aptamer-based detection systems:
  Single-domain antibodies (nanobodies) and aptamers offer several advantages over traditional antibodies, including smaller size (improving tissue penetration), higher stability, and more consistent performance. These alternative binding molecules can be conjugated with fluorophores, including alternatives to FITC with better photostability, providing improved options for PRDM16 detection in challenging contexts like thick tissue sections or in vivo imaging.

• CRISPR-based endogenous tagging:
  CRISPR/Cas9-mediated knock-in of fluorescent proteins or small epitope tags at the endogenous PRDM16 locus enables visualization of the protein under its native regulation without overexpression artifacts. When combined with live-cell imaging, this approach allows real-time tracking of PRDM16 dynamics during processes like stem cell differentiation or response to cellular stressors such as oxidative stress .

• Super-resolution microscopy with quantum dots:
  Conjugating PRDM16 antibodies to quantum dots instead of traditional fluorophores like FITC provides superior photostability and brightness. When combined with super-resolution microscopy techniques (STORM, PALM, STED), this approach enables visualization of PRDM16 localization at nanometer-scale resolution, potentially revealing previously undetectable subnuclear domains where PRDM16 concentrates to regulate specific gene sets.

• Mass cytometry (CyTOF):
  By conjugating PRDM16 antibodies to heavy metal isotopes instead of fluorophores, researchers can incorporate PRDM16 detection into high-dimensional CyTOF panels with 40+ parameters without fluorescence spectral overlap concerns. This technology is particularly valuable for comprehensive phenotyping of hematopoietic cells where PRDM16 plays regulatory roles in normal development and leukemogenesis .

• DNA-PAINT and Exchange-PAINT:
  These methods use transient binding of short fluorescently labeled DNA oligonucleotides to complementary strands attached to antibodies, enabling super-resolution imaging with effectively unlimited multiplexing capacity. For complex systems where PRDM16 interacts with multiple partners, this technology allows visualization of numerous proteins simultaneously without spectral overlap limitations.

These emerging technologies will likely transform PRDM16 research by providing higher resolution, more quantitative, and more comprehensive data on this protein's expression, localization, and function across different biological contexts and disease states.