PRDM16 Antibody, Biotin conjugated

What is PRDM16 and why is it important for research?

PRDM16 (PR domain zinc finger protein 16) is a transcription coregulator that controls the development of brown adipocytes in brown adipose tissue. While initially believed to be present only in brown adipose tissue, more recent studies have demonstrated that PRDM16 is also highly expressed in subcutaneous white adipose tissue. This zinc finger transcription factor plays a crucial role in determining cell fate between muscle and brown fat cells . At the molecular level, PRDM16 functions at the nuclear lamina (NL), where it mediates interactions between the genome and nuclear envelope, particularly affecting H3K9 methylation patterns . Its involvement in adipogenesis, metabolic regulation, and chromatin organization makes it a significant target for research in developmental biology, metabolic disorders, and epigenetic regulation.

What are the key specifications of commercially available PRDM16 Antibody (Biotin conjugated)?

Commercially available PRDM16 Antibody (Biotin conjugated) is typically a polyclonal antibody produced in rabbits. Key specifications include:

The antibody targets PRDM16, which has a calculated molecular weight of 140 kDa and is encoded by gene ID 63976 .

What is the difference between biotin-conjugated and unconjugated PRDM16 antibodies?

Biotin-conjugated PRDM16 antibodies have biotin molecules covalently attached to the antibody structure, while unconjugated antibodies lack this modification. The biotin conjugation offers several research advantages:

First, biotin-conjugated antibodies enable signal amplification through the strong affinity interaction between biotin and streptavidin/avidin detection systems. This amplification is particularly valuable when studying low-abundance proteins like PRDM16 in certain tissue types. The biotin-streptavidin system provides enhanced sensitivity compared to directly conjugated detection methods .

Second, biotin conjugation allows for greater flexibility in experimental design. Researchers can use the same biotinylated primary antibody with different streptavidin-conjugated reporters (fluorophores, enzymes, etc.) depending on their detection needs. This versatility is especially useful when developing multiplexed assays for PRDM16 detection alongside other proteins .

What are the optimal applications for PRDM16 Antibody (Biotin conjugated)?

PRDM16 Antibody (Biotin conjugated) is primarily optimized for Enzyme-Linked Immunosorbent Assay (ELISA) applications, where the biotin conjugation provides signal amplification advantages through streptavidin-based detection systems . While ELISA represents the validated application, researchers should note that other applications may require optimization.

For alternative applications such as Western Blotting, Immunohistochemistry, or Immunofluorescence, unconjugated PRDM16 antibodies have demonstrated effectiveness with the following recommended dilutions:

• Western Blot (WB): 1:2000-1:10000

• Immunohistochemistry (IHC): 1:50-1:500

• Immunofluorescence (IF)/ICC: 1:125-1:500

When specifically investigating PRDM16's localization at the nuclear periphery or its interactions with other proteins like GLP and LaminB, techniques such as Proximity Ligation Assay (PLA) and co-immunoprecipitation (co-IP) have proven effective in research settings . These more specialized techniques may require additional optimization when using biotin-conjugated antibodies.

How should researchers design co-immunoprecipitation experiments to study PRDM16 interactions?

When designing co-immunoprecipitation (co-IP) experiments to study PRDM16 interactions with proteins like GLP or LaminB, researchers should follow this methodological approach:

First, prepare nuclear extracts from cells expressing PRDM16 (such as fibro-adipogenic progenitors or brown adipose tissue) by lysing cells in appropriate buffer containing protease inhibitors, followed by nuclear isolation and extraction of nuclear proteins. Clear the lysates by centrifugation (typically 14,000g for 20 minutes at 4°C) and pre-clear with Protein A/G magnetic beads for 2 hours at 4°C to reduce non-specific binding .

For the immunoprecipitation step, use 1-2 mg of nuclear extract with 5-15 μg of anti-PRDM16 antibody (for PRDM16 pulldown) or anti-LaminB antibody (for LaminB pulldown). Include appropriate IgG controls to assess non-specific binding. Incubate overnight at 4°C with gentle rotation .

After incubation, add Protein A/G beads for 2 hours to recover immunocomplexes, then wash extensively (at least six washes) with IP buffer containing 50 mM Tris (pH 7.9), 150 mM NaCl, 1 mM EDTA, and 1 mM EGTA. Elute immunocomplexes with Laemmli sample buffer and heat at 95°C before proceeding to SDS-PAGE and Western blotting .

When probing for interacting partners, consider examining G9a, GLP, and LaminB, which have demonstrated interactions with PRDM16 at the nuclear periphery. Use Western blotting with appropriate antibodies to detect these interacting proteins in your immunoprecipitated samples.

What methods can be used to study PRDM16 localization at the nuclear lamina?

To investigate PRDM16 localization at the nuclear lamina, researchers can employ several complementary techniques:

Proximity Ligation Assay (PLA) can be used to visualize and quantify protein-protein interactions between PRDM16 and nuclear lamina components like LaminB or other interacting partners such as GLP. This technique generates fluorescent dots when two proteins of interest are within 40 nm of each other. PLA has successfully demonstrated that PRDM16 and GLP interact preferentially at the nuclear periphery . When analyzing PLA results, quantify the number of interacting dots in contact with nuclear edges to assess peripheral localization.

Confocal microscopy with immunofluorescence staining can directly visualize PRDM16 colocalization with nuclear lamina markers. Double immunostaining for PRDM16 and LaminB followed by confocal microscopy analysis has revealed their colocalization at the nuclear periphery . When conducting these experiments, ensure proper controls for antibody specificity, particularly when using biotin-conjugated antibodies in multi-color imaging approaches.

For genome-wide analysis of PRDM16's role at the nuclear lamina, Chromatin Immunoprecipitation sequencing (ChIP-seq) for LaminB and H3K9me2 in control versus PRDM16-depleted cells can identify genomic regions associated with the nuclear lamina in a PRDM16-dependent manner. These approaches have revealed that PRDM16 mediates interactions between the genome and nuclear lamina, particularly affecting H3K9me2-enriched heterochromatin domains .

How does PRDM16 depletion affect nuclear lamina-associated domains (LADs) and H3K9 methylation patterns?

PRDM16 depletion significantly disrupts nuclear lamina-associated domains (LADs) and H3K9 methylation patterns, revealing its crucial role in organizing heterochromatin at the nuclear periphery.

LaminB ChIP-seq analyses in control versus PRDM16-depleted fibro-adipogenic progenitor (FAP) cells demonstrated that PRDM16 loss dramatically reduces genome-nuclear lamina interactions. Control cells exhibit approximately 26.14% LaminB occupancy genome-wide (corresponding to 3007 targeted genes), while PRDM16-depleted cells show a marked reduction to 12.46% occupancy (only 1680 targeted genes). Additionally, the remaining LADs in PRDM16-depleted cells are narrower compared to control cells .

Regarding H3K9 methylation, PRDM16 depletion causes redistribution of H3K9me2 marks from the nuclear periphery to the nuclear interior. In control cells, H3K9me2 sharply marks a peripheral heterochromatic layer, while PRDM16-depleted cells show diffuse H3K9me2 distribution throughout the nucleus. ChIP-seq analysis revealed that control FAPs have approximately 19.8% genome-wide H3K9me2 occupancy, which decreases to 13.75% upon PRDM16 depletion, with narrower H3K9me2-enriched domains .

This redistribution has functional consequences on gene expression. Genes losing H3K9me2 upon PRDM16 depletion tend to be upregulated, while genes gaining H3K9me2 are typically downregulated. These findings position PRDM16 as a critical tethering factor that maintains H3K9me2-marked heterochromatin at the nuclear lamina, affecting both chromatin organization and gene expression programs .

What is the functional relationship between PRDM16 and the G9a/GLP methyltransferase complex?

The functional relationship between PRDM16 and the G9a/GLP methyltransferase complex represents a sophisticated mechanism for controlling chromatin organization and gene expression at the nuclear periphery.

PRDM16 physically interacts with both G9a and GLP, as demonstrated by proximity ligation assay (PLA) and co-immunoprecipitation studies. Importantly, the PRDM16-GLP interaction shows a distinctive localization pattern, with interacting dots predominantly positioned toward the nuclear periphery. Quantitative analysis confirmed that PRDM16-GLP interactions occur significantly more often at nuclear edges compared to G9a-GLP interactions, which are more prevalent in the nuclear interior .

This peripheral interaction is functionally significant, as PRDM16 mediates the recruitment of G9a/GLP to the nuclear lamina, where the methyltransferase complex deposits H3K9me2 marks on peripheral heterochromatin. When PRDM16 is depleted, the interaction between GLP and the nuclear periphery is abolished, resulting in redistribution of H3K9me2 from the nuclear periphery to the nuclear interior .

Mechanistically, PRDM16 serves as a bridge between the nuclear lamina (through its interaction with LaminB) and the G9a/GLP complex, facilitating H3K9me2 deposition at lamina-associated domains. This coordinated action helps establish and maintain repressive heterochromatin domains at the nuclear periphery, contributing to proper gene silencing and nuclear architecture .

How can researchers distinguish between PRDM16's roles as a transcriptional repressor versus activator?

Distinguishing between PRDM16's dual roles as both a transcriptional repressor and activator requires carefully designed experiments that integrate multiple molecular approaches.

To identify repressive functions, researchers should investigate PRDM16's interactions with heterochromatin-associated factors and histone modifiers. Co-immunoprecipitation experiments followed by mass spectrometry can reveal interactions with repressive complexes like G9a/GLP. ChIP-seq for PRDM16, H3K9me2, and LaminB in the same cellular context can identify genomic regions where PRDM16 colocalizes with repressive histone marks and nuclear lamina association. RNA-seq analysis comparing wildtype and PRDM16-depleted cells can then correlate these repressive chromatin features with transcriptional outcomes .

For PRDM16's activating functions, researchers should examine its interactions with the Mediator complex, which has been documented in previous studies. PRDM16 can act as a transcriptional activator through interaction with Mediator components, influencing gene expression in brown adipose tissue and other contexts. ChIP-seq for PRDM16 and activating histone marks (H3K4me3, H3K27ac) can identify regions where PRDM16 associates with active chromatin .

Interestingly, these seemingly contradictory functions may be reconciled through a "tug-of-war" mechanism. While PRDM16 tethers certain genomic regions to the repressive environment at the nuclear periphery, this repositioning may simultaneously relocate other genomic regions to the transcriptionally active nuclear interior. This model explains how PRDM16 can simultaneously repress certain genes while activating others in the same cell .

What are common issues when using PRDM16 Antibody (Biotin conjugated) in ELISA and how can they be addressed?

When using PRDM16 Antibody (Biotin conjugated) in ELISA applications, researchers may encounter several technical challenges that require specific troubleshooting approaches:

High background signal is a common issue that can result from several factors. Endogenous biotin in samples, particularly those derived from biotin-rich tissues like liver or kidney, can interfere with detection. To address this, researchers should block endogenous biotin using streptavidin/biotin blocking kits before antibody application. Non-specific binding can also contribute to background; optimize blocking conditions using different blockers (BSA, casein, or commercial blocking buffers) and include 0.05-0.1% Tween-20 in wash buffers .

Low or no signal detection may occur despite the presence of target protein. In such cases, verify PRDM16 expression in your sample type first, as expression levels vary significantly between tissues (higher in brown adipose tissue, detectable in subcutaneous white adipose tissue). For low-abundance samples, consider signal amplification using additional layers of streptavidin-HRP or streptavidin-poly-HRP systems. Antibody concentration should be titrated; despite the high purity (>95%) of commercial preparations, optimal dilutions should be determined empirically for each experimental system .

Poor reproducibility between experiments often stems from inconsistent handling. PRDM16 antibody requires storage at -20°C with minimal freeze-thaw cycles. Aliquot the antibody upon receipt to avoid repeated freeze-thaw cycles, which can decrease activity. Light exposure should be minimized for biotin-conjugated antibodies. When preparing working dilutions, use fresh diluent containing carrier protein (0.1% BSA) to prevent adsorption to tube walls .

How can researchers validate the specificity of PRDM16 Antibody (Biotin conjugated)?

Validating the specificity of PRDM16 Antibody (Biotin conjugated) is critical for ensuring reliable research results. A comprehensive validation approach should include:

Positive and negative control samples are essential for antibody validation. For positive controls, use tissues or cell lines known to express PRDM16, such as brown adipose tissue, subcutaneous white adipose tissue, or cell lines like U2OS or K-562 which have been confirmed to express PRDM16 . For negative controls, consider using tissues with minimal PRDM16 expression or, ideally, PRDM16 knockout/knockdown samples where the target protein has been depleted.

Western blot analysis can confirm antibody specificity by demonstrating a single band at the expected molecular weight (~140 kDa for PRDM16). Perform side-by-side comparison of biotin-conjugated and unconjugated antibodies against the same samples to ensure consistent detection patterns. Include PRDM16-depleted samples to confirm absence of the specific band .

Immunoprecipitation followed by mass spectrometry provides rigorous validation by confirming that the antibody pulls down PRDM16 and known interacting partners such as G9a, GLP, and LaminB. This approach helps verify that the antibody recognizes the native protein in solution and can effectively immunoprecipitate PRDM16 complexes .

Cross-reactivity testing should be performed against related PR domain-containing proteins (other PRDM family members) to ensure the antibody specifically recognizes PRDM16 without detecting closely related proteins. This is particularly important when studying tissues that express multiple PRDM family members.

What storage and handling precautions should be taken to maintain PRDM16 Antibody (Biotin conjugated) activity?

Proper storage and handling of PRDM16 Antibody (Biotin conjugated) is crucial for maintaining its activity and ensuring consistent experimental results:

Temperature management is critical for antibody stability. Store PRDM16 Antibody (Biotin conjugated) at -20°C in a non-frost-free freezer to avoid temperature fluctuations. The antibody is typically supplied in a buffer containing 50% glycerol, which prevents freezing solid at -20°C and allows for direct pipetting without complete thawing .

Aliquoting upon receipt is strongly recommended to avoid repeated freeze-thaw cycles, which can cause antibody degradation and reduced activity. Prepare small single-use aliquots based on your typical experimental needs. When making aliquots, use sterile techniques and sterile tubes to prevent microbial contamination .

Light protection is essential for biotin-conjugated antibodies, as exposure to light can cause photobleaching of the biotin moiety and reduce conjugate stability. Store aliquots in amber tubes or wrap containers in aluminum foil. When working with the antibody, minimize exposure to direct light and work under reduced ambient lighting conditions when possible .

Buffer composition affects antibody stability during storage and use. PRDM16 Antibody (Biotin conjugated) is typically supplied in PBS with preservatives like 0.03% Proclin-300 or 0.02% sodium azide and 50% glycerol at pH 7.4. When preparing working dilutions, use fresh buffer containing a carrier protein (0.1-0.5% BSA) to prevent adsorption to tube walls. Working dilutions should be prepared just before use and not stored for extended periods .

How should researchers interpret unexpected molecular weight variations in PRDM16 Western blots?

When researchers encounter unexpected molecular weight variations in PRDM16 Western blots, careful analysis and consideration of several factors is necessary for accurate interpretation:

• Isoforms and splice variants: PRDM16 has multiple isoforms resulting from alternative splicing. The UniProt database lists several secondary accession numbers (A6NHQ8, B1AJP7, B1AJP8, B1AJP9, B1WB48, Q8WYJ9, Q9C0I8) associated with PRDM16, suggesting the existence of multiple protein variants . These isoforms may have different molecular weights and functional properties.

• Post-translational modifications: PRDM16 can undergo various post-translational modifications, including phosphorylation, which may alter its apparent molecular weight in SDS-PAGE. These modifications can be tissue or context-specific and may reflect different functional states of the protein.

• Proteolytic processing: PRDM16 may undergo proteolytic cleavage in certain cellular contexts, generating fragments that retain immunoreactivity but appear at lower molecular weights.

To distinguish between these possibilities, researchers should:

• Compare results across multiple tissue/cell types to identify consistent patterns

• Use multiple antibodies targeting different epitopes of PRDM16

• Perform validation using PRDM16 overexpression and knockdown/knockout systems

• Consider isoform-specific PCR to correlate protein observations with transcript expression

When reporting results with molecular weight variations, clearly document the observed pattern and discuss possible interpretations rather than dismissing unexpected bands as non-specific .

What considerations are important when analyzing PRDM16 localization using immunofluorescence?

When analyzing PRDM16 localization using immunofluorescence, researchers should consider several technical and biological factors to ensure accurate interpretation:

When examining nuclear periphery localization, confocal microscopy with z-stack acquisition is essential. Single optical sections can be misleading when analyzing nuclear periphery proteins. Collect z-stacks spanning the entire nucleus and analyze multiple sections or generate maximum intensity projections. For quantitative analysis of PRDM16 nuclear localization patterns, measure fluorescence intensity from the nuclear periphery to the center using line profile analysis across multiple cells .

Consider dual or triple staining approaches to contextualize PRDM16 localization. Co-staining with LaminB provides nuclear envelope reference, while H3K9me2 staining can confirm the relationship between PRDM16 and heterochromatin domains. When using biotin-conjugated primary antibodies, carefully design your staining protocol to avoid cross-reactivity with other detection systems .

Finally, be aware that PRDM16 exhibits both nuclear and cytoplasmic localization in some cell types. The cytoplasmic signal is not an artifact but reflects PRDM16's reported cytosolic function as an H3K9 monomethyltransferase on free histone H3. Document both nuclear and cytoplasmic patterns when present .

How can researchers integrate ChIP-seq and RNA-seq data to understand PRDM16's genomic functions?

Integrating ChIP-seq and RNA-seq data provides powerful insights into PRDM16's genomic functions, particularly its dual roles in gene regulation. A comprehensive analytical approach should include:

First, perform ChIP-seq targeting PRDM16, H3K9me2, and LaminB in your experimental system, along with RNA-seq on matched samples. For differential analysis, compare wildtype conditions with PRDM16 depletion (knockout or knockdown). Process ChIP-seq data using standardized pipelines to identify enriched regions, then use tools like enriched domain detector (EDD) to define domains rather than focusing solely on peaks, as PRDM16 often functions in broad domains .

For genomic integration analysis, overlay PRDM16, H3K9me2, and LaminB ChIP-seq datasets to identify regions where these features coincide, which represent candidate PRDM16-dependent heterochromatic domains at the nuclear periphery. Calculate the percentage of genome coverage for each mark, noting that typically ~20% of the genome is marked by H3K9me2 in control conditions, with significant reduction upon PRDM16 depletion .

For advanced analysis, consider chromosome conformation capture techniques (Hi-C, 4C) to determine how PRDM16 influences three-dimensional genome organization. The "tug-of-war" model suggests that PRDM16-mediated tethering of certain regions to the nuclear periphery may simultaneously relocate other regions to transcriptionally active compartments, explaining PRDM16's dual repressive and activating functions .