PRDM16 Antibody, HRP conjugated

What is PRDM16 and why is it significant in research?

PRDM16 (PR domain containing 16) is a zinc finger transcription factor that functions as a critical transcription coregulator controlling multiple developmental and metabolic pathways. The protein has a calculated molecular weight of 140.3 kDa and plays essential roles in:

• Brown adipocyte development in brown adipose tissue

• Ventricular cardiomyocyte identity specification

• Cell fate determination between muscle and brown fat cells

• Epigenetic regulation through histone H3K9 monomethylation (H3K9me1)

Recent research has revealed that contrary to earlier beliefs that PRDM16 was exclusively expressed in brown adipose tissue, it is also highly expressed in subcutaneous white adipose tissue, expanding its research significance . The PRDM16 marker can also be used to identify Gray Matter Splatter Neurons according to the HuBMAP Human Reference Atlas v1.4 .

What advantages does HRP conjugation provide for PRDM16 antibody applications?

HRP (Horseradish Peroxidase) conjugation offers several methodological advantages that enhance research workflows:

The HRP conjugation allows direct binding to PRDM16 antigens, which significantly simplifies ELISA workflows by eliminating the need for secondary antibodies. This design reduces assay time and minimizes potential cross-reactivity issues that can compromise experimental results.

What are the typical applications for PRDM16 antibody, HRP conjugated?

• Western Blotting (WB)

• Immunohistochemistry (IHC)

• Immunofluorescence (IF)

Researchers should note that while the antibody has been validated for human samples, specific reactivity depends on the antibody clone. Some PRDM16 antibodies demonstrate cross-reactivity with mouse and rat samples, expanding their utility in comparative studies across species .

How should optimization of HRP-conjugated PRDM16 antibody dilutions be approached for different applications?

Optimization requires systematic titration across multiple dilution ranges. For HRP-conjugated PRDM16 antibodies, I recommend the following methodological approach:

For ELISA applications:

• Begin with a broad range titration (1:500, 1:1000, 1:2000, 1:5000, 1:10000)

• Perform a narrow range titration around the best performing dilution

• Include positive and negative controls with each titration series

• Plot signal-to-noise ratios against antibody concentration to determine optimal dilution

The manufacturer's guidance indicates that optimal working dilution should be determined experimentally for each specific application . This is essential as optimal dilutions vary significantly between applications:

• For Western Blotting: Published data suggests dilutions ranging from 1:2000-1:10000

• For Immunohistochemistry: More concentrated dilutions (1:50-1:500) are typically required

• For Immunofluorescence: Intermediate dilutions (1:125-1:500) are commonly effective

Sample-dependent variables should be carefully considered during optimization, particularly when working with tissues expressing variable levels of PRDM16.

What protocol modifications are necessary when transitioning from unconjugated to HRP-conjugated PRDM16 antibody in ELISA?

When transitioning to HRP-conjugated PRDM16 antibody for ELISA, implement these methodological adjustments:

• Eliminate secondary antibody incubation step entirely

• Reduce total protocol time by approximately 1-2 hours

• Adjust substrate incubation time (typically shorter for direct detection)

• Modify washing steps (usually fewer washes required)

• Re-optimize antibody concentration (HRP-conjugated antibodies often require different optimal concentrations than unconjugated versions)

These modifications leverage the direct detection capability of HRP conjugation, which allows for simplified workflows while maintaining or improving sensitivity.

What are the optimal storage conditions for maintaining HRP-conjugated PRDM16 antibody activity?

To maintain maximum enzymatic activity of the HRP conjugate while preserving antibody binding specificity:

• Store at -20°C in aliquots to minimize freeze-thaw cycles

• Use storage buffer containing PBS with 0.02% sodium azide and 50% glycerol at pH 7.3

• Ensure stable temperature during storage (temperature fluctuations accelerate activity loss)

• Aliquot upon receipt to prevent repeated freeze-thaw cycles

• For long-term storage (>1 year), consider -80°C storage

Most HRP-conjugated antibodies remain stable for one year after shipment when stored properly. For small volume formats (20μl), some preparations may contain 0.1% BSA as a stabilizer .

How can HRP-conjugated PRDM16 antibody be utilized to investigate brown adipose tissue differentiation?

PRDM16 plays a crucial role in brown adipose tissue (BAT) development and function. To leverage HRP-conjugated PRDM16 antibody for BAT differentiation studies:

• Time-course analysis: Monitor PRDM16 expression levels during differentiation of preadipocytes using quantitative ELISA

• Co-localization studies: Combine with other markers (UCP1, PGC-1α) to characterize differentiation stages

• Comparative analysis: Evaluate PRDM16 expression across different adipose tissue depots (brown, beige, white)

• Intervention studies: Assess changes in PRDM16 levels following treatment with factors known to promote browning (cold exposure, β3-adrenergic agonists)

When performing immunohistochemistry on adipose tissue, it's recommended to use antigen retrieval with TE buffer at pH 9.0 to maximize epitope accessibility. Alternatively, citrate buffer at pH 6.0 may be used, though this might yield different staining intensities .

What strategies can overcome potential cross-reactivity issues when studying PRDM16 in complex tissue samples?

When investigating PRDM16 in heterogeneous tissues containing multiple cell types, several advanced strategies can minimize cross-reactivity concerns:

• Peptide competition assays: Pre-incubate antibody with immunizing peptide to confirm specificity

• Knockout/knockdown validation: Use PRDM16-null or PRDM16-depleted samples as negative controls

• Epitope mapping: Select antibodies targeting unique epitopes (such as AA 448-683) with minimal homology to related proteins

• Multi-antibody approach: Use antibodies targeting different epitopes to confirm consistent staining patterns

• Cell sorting: Enrich for target populations before antibody application

For particularly challenging tissue samples like cardiac tissue, where PRDM16 has been implicated in ventricular cardiomyocyte identity specification, consider using more sensitive detection methods coupled with careful optimization of antigen retrieval conditions.

How can researchers quantitatively analyze PRDM16 expression levels in relation to metabolic disease models?

For quantitative analysis of PRDM16 expression in metabolic disease models, implement this systematic workflow:

• Standard curve generation: Create a standard curve using recombinant PRDM16 protein for absolute quantification

• Normalization strategy: Select appropriate housekeeping proteins that remain stable in metabolic disease states

• Tissue-specific extraction optimization: Modify protein extraction protocols to account for high lipid content in adipose samples

• Statistical approach: Apply multivariate analysis to correlate PRDM16 levels with metabolic parameters

• Dynamic range verification: Ensure detection system provides sufficient dynamic range to capture disease-related changes

When comparing PRDM16 expression between healthy and diseased states, it's critical to maintain consistent sampling procedures, particularly for adipose tissue, where regional heterogeneity can significantly impact results .

What are common causes of reduced signal when using HRP-conjugated PRDM16 antibody, and how can they be addressed?

When encountering diminished signal with HRP-conjugated PRDM16 antibody, systematically evaluate these potential causes and solutions:

For optimal results with HRP-conjugated antibodies, ensure substrate compatibility and always include positive control samples with known PRDM16 expression, such as U2OS cells or K-562 cells, which have been validated for PRDM16 detection .

How should researchers address variability in PRDM16 detection across different cell lines and tissue types?

Addressing variability in PRDM16 detection requires a methodical approach tailored to sample characteristics:

When comparing PRDM16 expression across tissues, it's essential to optimize each tissue type independently rather than applying a single standardized protocol across all samples.

What quality control measures should be implemented when validating a new lot of HRP-conjugated PRDM16 antibody?

Implementing a comprehensive quality control workflow for new antibody lots is essential for research reproducibility:

• Comparative analysis with previous lot:

  • Run side-by-side Western blots with identical samples

  • Compare EC50 values in ELISA assays

  • Assess background levels in parallel staining procedures

• Specificity verification:

  • Confirm expected molecular weight (~140 kDa)

  • Verify staining patterns match known PRDM16 distribution

  • Perform peptide competition assays

• Functionality testing:

  • Verify HRP activity using standard substrates

  • Assess detection limits using dilution series

  • Confirm signal stability over time

• Cross-platform validation:

  • Test across multiple applications (ELISA, WB, IHC as appropriate)

  • Verify reactivity with expected species (human samples)

  • Document all validation parameters for reference

Proper validation is particularly important for advanced research applications investigating PRDM16's role in critical processes like cardiac development or adipogenesis.

How can HRP-conjugated PRDM16 antibody contribute to research on cardiac development and disease?

Recent research has established PRDM16's crucial role in cardiac development, offering new research directions for HRP-conjugated PRDM16 antibody applications:

• Ventricular identity specification:

  • Quantify PRDM16 expression in developing cardiomyocytes

  • Correlate expression levels with ventricular versus atrial marker expression

  • Monitor changes in PRDM16 levels during cardiac differentiation protocols

• Cardiac disease models:

  • Investigate PRDM16 expression in cardiomyopathy models

  • Evaluate changes in subcellular localization during cardiac stress

  • Correlate PRDM16 levels with functional cardiac parameters

• Therapeutic development:

  • Screen compounds that modulate PRDM16 expression in cardiac tissues

  • Monitor PRDM16 as a biomarker for cardiac regeneration approaches

  • Track PRDM16 levels during stem cell-based cardiac therapies

PRDM16 has been specifically implicated in ventricular cardiomyocyte identity specification, with deletion leading to cardiac dysfunction in mouse models. This suggests potential applications in studying congenital heart defects and acquired cardiomyopathies.

What novel methodologies are being developed to study PRDM16's epigenetic functions using HRP-conjugated antibodies?

Emerging research on PRDM16's epigenetic activities has opened new methodological approaches:

• ChIP-sequencing adaptations:

  • Use HRP-conjugated PRDM16 antibodies for streamlined ChIP protocols

  • Develop sequential ChIP methods to analyze PRDM16 co-occupancy with histone marks

  • Implement CUT&RUN or CUT&Tag protocols for improved resolution of binding sites

• Histone modification analysis:

  • Correlate PRDM16 binding with H3K9me1 levels through dual staining approaches

  • Develop quantitative assays for measuring PRDM16 methyltransferase activity

  • Map genome-wide changes in chromatin accessibility following PRDM16 manipulation

• Protein interaction studies:

  • Adapt proximity ligation assays using HRP-conjugated antibodies

  • Develop co-immunoprecipitation protocols optimized for chromatin-associated complexes

  • Implement BioID or APEX2 proximity labeling to map PRDM16 interaction networks

PRDM16's function as a histone methyltransferase that monomethylates H3K9 positions it as a critical epigenetic regulator, influencing chromatin structure and gene expression programs that control cell fate decisions.

How can researchers integrate HRP-conjugated PRDM16 antibody into multiparametric analysis of energy metabolism?

Advanced integration of PRDM16 detection into comprehensive metabolic research requires sophisticated methodological approaches:

• Multiplexed tissue analysis:

  • Combine HRP-conjugated PRDM16 antibody with fluorescent detection of metabolic markers

  • Develop sequential staining protocols to maximize parameter detection

  • Implement computational image analysis for quantitative co-expression studies

• Systems biology integration:

  • Correlate PRDM16 levels with transcriptomic profiles of metabolic genes

  • Develop protocols for single-cell analysis of PRDM16 expression in heterogeneous tissues

  • Create mathematical models incorporating PRDM16 regulatory networks

• Functional metabolic correlation:

  • Design protocols linking PRDM16 detection with real-time metabolic measurements

  • Develop ex vivo systems for manipulating PRDM16 while monitoring energy expenditure

  • Implement longitudinal studies correlating PRDM16 expression with metabolic adaptation

PRDM16's established role in promoting brown adipose tissue differentiation and energy expenditure makes it a valuable target for obesity research, metabolic syndrome studies, and investigations into adaptive thermogenesis mechanisms .