PRDM16 Antibody

What is PRDM16 and why are specific antibodies needed for its detection?

PRDM16 is a zinc finger transcription factor containing an N-terminal PR domain that functions as a transcriptional regulator in multiple tissues . The protein has a molecular weight of approximately 140-170 kDa and plays crucial roles in:

• Brown adipose tissue differentiation and function

• Ventricular cardiomyocyte specification

• BMP and TGF-β signaling pathways

• The pathogenesis of myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML)

Specific antibodies are essential because PRDM16 exists in multiple isoforms (including a truncated form in certain leukemias), and its expression is highly tissue-specific, requiring reagents with validated specificity for accurate detection .

A methodical validation approach should include:

• Positive controls: Use K562 human chronic myelogenous leukemia cells , brown adipose tissue , or HEK-293 cells with confirmed PRDM16 expression

• Negative controls: Include PRDM16 knockout samples when available

• Western blot verification: Confirm a specific band at the expected molecular weight of ~140-170 kDa

• Cross-reactivity testing: If working with animal models, verify species cross-reactivity as many PRDM16 antibodies work across human, mouse, and rat samples

• Application-specific validation: For IHC/IF, include antigen retrieval optimization (pH 9.0 TE buffer or pH 6.0 citrate buffer)

How can I optimize PRDM16 chromatin immunoprecipitation experiments?

Based on successful PRDM16 ChIP and CUT&TAG studies , consider these methodological approaches:

• Crosslinking protocol: Use dual crosslinking with DSG (disuccinimidyl glutarate) followed by formaldehyde to stabilize protein-protein interactions before capturing DNA binding

• Sonication optimization: Target 200-500bp fragments for optimal resolution

• Antibody selection: For PRDM16 CUT&TAG experiments, validated antibodies with low background are essential, as shown in neural stem cell studies

• Controls: Include IgG controls and PRDM16 knockout samples to determine specificity

• Sequential ChIP considerations: For co-occupancy studies with partners like SMAD4, optimize protocols for each antibody separately before sequential ChIP

A typical workflow for studying PRDM16 genomic binding includes:

• Primary ChIP with PRDM16 antibody

• Secondary ChIP with partner protein antibodies (e.g., SMAD4)

• qPCR validation of enrichment at known target genes such as Wnt7b and Id1

What methodological approaches are recommended for studying PRDM16 protein stability?

To investigate PRDM16 protein stability and degradation mechanisms, cycloheximide chase assays have proven effective :

• Experimental design:

  • Treat differentiated adipocytes with cycloheximide at 20 µg/ml

  • Treat transfected HEK293T cells with cycloheximide at 10 µg/ml

  • Collect samples at specific time points (e.g., 0, 2, 4, 8 hours)

• Analysis protocol:

  • Perform western blotting with PRDM16 antibody

  • Normalize band intensity to β-actin

  • Plot relative protein levels vs. time to determine half-life

  • Compare between experimental conditions (e.g., wild-type vs. mutant)

• Additional considerations:

  • Include proteasome inhibitors (MG132) in parallel experiments to confirm degradation pathway

  • For ubiquitination studies, use in vitro ubiquitination assays with methyl-Ub and immunoprecipitate with PRDM16 antibody

How can PRDM16 antibodies be used to investigate tissue-specific functions in brown adipose tissue?

For investigating PRDM16's role in brown adipose tissue (BAT), consider these methodological approaches:

• Tissue preparation:

  • For IHC: Prepare frozen sections with optimal fixation (4% PFA)

  • For protein analysis: Rapidly harvest and freeze BAT to prevent protein degradation

• Functional assays:

  • Compare PRDM16 binding across BAT-selective genes using ChIP-qPCR

  • Analyze recruitment of MED1 (Mediator complex component) to PRDM16 target sites

  • Examine chromatin architecture changes at BAT-selective genes like Ppargc1a and Pparα

• Expression analysis techniques:

  • Use PRDM16 antibodies to compare protein levels between white adipose tissue and BAT

  • Combine with RNA Polymerase II ChIP to correlate PRDM16 binding with transcriptional activity

  • Assess H3K27 acetylation levels at PRDM16 binding sites to evaluate enhancer activity

PRDM16 is critical for maintaining brown adipocyte identity, with knockout studies showing dramatic reductions in thermogenic gene expression (90-95% reduction in Ucp1, Cidea, and Dio2) .

What are the techniques for analyzing PRDM16 post-translational modifications?

To investigate PRDM16 post-translational modifications (PTMs), particularly ubiquitination:

• Sample preparation for ubiquitination analysis:

  • Purify recombinant Flag-PRDM16 protein

  • Perform in vitro ubiquitination reactions using methyl-Ub

  • Separate by SDS-PAGE and stain with Coomassie blue

  • Excise protein bands for mass spectrometry analysis

• Mass spectrometry protocol:

  • Perform in-gel digestion of excised bands

  • Extract peptides with 5% formic acid/50% acetonitrile

  • Resuspend in 2%/0.1% acetonitrile/formic acid solution

  • Separate on an analytical capillary column with C18-reversed-phase silica beads

  • Employ HPLC gradient (5-35% in 60 min) for optimal separation

  • Analyze using LTQ Orbitrap Velos Pro mass spectrometer

• Two-step immunoprecipitation for ubiquitination detection:

  • First-round: Immunoprecipitate with anti-Flag M2 affinity gel

  • Denature bound proteins by boiling in 1% SDS

  • Dilute 1:10 and re-immunoprecipitate

  • Analyze by immunoblotting for ubiquitin and PRDM16

How can I differentiate between full-length and truncated PRDM16 in leukemia research?

The truncated form of PRDM16 (lacking the PR domain) is associated with certain leukemias through the t(1;3)(p36;q21) translocation . To distinguish between isoforms:

• Antibody selection strategy:

  • Use antibodies targeting different regions of PRDM16

  • N-terminal antibodies will detect full-length but not some truncated forms

  • C-terminal antibodies will detect both full-length and truncated variants

• Western blot analysis:

  • The full-length PRDM16 appears at approximately 170 kDa

  • Truncated forms will appear at lower molecular weights

  • Use positive control K562 cells that express PRDM16

• Functional validation approaches:

  • Compare DNA binding patterns using ChIP-seq

  • Assess differential protein interactions through co-immunoprecipitation

  • Evaluate transcriptional effects on target genes to distinguish functional differences

What are the optimal conditions for PRDM16 antibody usage in Western blotting?

For optimal Western blot detection of PRDM16:

• Sample preparation:

  • Use reducing conditions for sample preparation

  • Include protease inhibitors to prevent degradation

  • For adipose tissue samples, specific lysis buffers may be required to handle high lipid content

• Protocol optimizations:

  • Use PVDF membrane for better protein retention

  • A specific band for PRDM16 should be detected at approximately 140-170 kDa

  • For K562 cell lysates, use 1 µg/mL antibody concentration

  • Use HRP-conjugated secondary antibodies for standard detection

• Buffer considerations:

  • Use Immunoblot Buffer Group 8 for optimal results with certain antibodies

  • Consider using milk-free blocking buffers if background is high

How should I optimize immunohistochemistry protocols for PRDM16 detection in tissues?

For effective IHC/IF detection of PRDM16 in tissue samples:

• Tissue preparation:

  • For frozen sections: Use immersion fixation

  • For paraffin sections: Optimize antigen retrieval (pH 9.0 TE buffer or pH 6.0 citrate buffer)

• Antibody incubation parameters:

  • For frozen mouse embryo sections: 10 µg/mL antibody concentration

  • Overnight incubation at 4°C provides optimal results

  • Anti-sheep secondary antibodies conjugated to fluorophores (e.g., NorthernLights 557) work well for PRDM16 detection

• Tissue-specific considerations:

  • Brown adipose tissue shows high PRDM16 expression

  • In mouse embryos, specific staining is localized to the trigeminal ganglion

  • For cardiac tissue, ventricular myocardium shows PRDM16 expression, particularly in the compact layer

• Visualization and counterstaining:

  • DAPI counterstaining helps visualize nuclear localization

  • Use appropriate filters for fluorescently labeled secondary antibodies

  • Include controls (PRDM16 knockout tissue) whenever possible

What approaches are effective for using PRDM16 antibodies in single-cell studies?

For applying PRDM16 antibodies in single-cell research contexts:

• Fluorescence-activated cell sorting preparation:

  • Use reporter systems (e.g., Rosa26-tdTomato) to label PRDM16-expressing cells

  • FACS-purify cells before single-cell analysis

• Single-cell immunofluorescence optimization:

  • Use newer generation super-resolution microscopy for detailed subcellular localization

  • Optimize fixation protocols to preserve epitope accessibility

  • Consider tyramide signal amplification for low abundance detection

• Single-cell RNA-seq integration:

  • Correlate protein levels (by antibody staining) with transcriptome data

  • For cardiac studies, PRDM16 antibodies can help identify compact myocardium-specific cells

  • Unsupervised clustering can separate cell populations based on PRDM16 expression

This approach has been successfully used to identify PRDM16-expressing cardiomyocyte populations (Nkx2-5+) distinct from epicardial cells (Tcf21+) and atrioventricular cushion mesenchyme cells (Postn+) .

How can I address non-specific binding issues with PRDM16 antibodies?

When encountering non-specific binding:

• Validation approaches:

  • Compare multiple antibodies targeting different PRDM16 epitopes

  • Include knockout or knockdown controls whenever possible

  • Test on cell lines with confirmed expression versus non-expressing lines

• Protocol optimizations:

  • Increase blocking stringency (5% BSA or specialized blocking reagents)

  • Optimize antibody concentration through titration experiments

  • For Western blot, extend washing steps and use additives like 0.1% SDS in TBST

• Application-specific considerations:

  • For ChIP assays, include pre-clearing steps with protein A/G beads

  • For IHC, optimize antigen retrieval conditions and test multiple fixation methods

  • For IP experiments, consider two-step approaches to reduce non-specific interactions

What strategies can address inconsistent PRDM16 detection across experiments?

For researchers experiencing inconsistent detection:

• Sample handling considerations:

  • PRDM16 may be subject to degradation; use fresh samples and include protease inhibitors

  • Standardize sample collection and processing protocols

  • For adipose tissue, control for developmental stage and environmental conditions (temperature)

• Experimental standardization:

  • Maintain consistent antibody lots when possible

  • Include internal loading controls appropriate for the experimental context

  • Standardize protein quantification methods before immunoblotting

• Tissue-specific optimizations:

  • For brown adipose tissue: Control for temperature adaptation status of animals

  • For cardiac samples: Consider developmental stage carefully, as PRDM16 expression changes during development

  • For cell culture: Standardize differentiation protocols as PRDM16 levels may fluctuate with differentiation state

How can I detect low abundance PRDM16 in certain tissue contexts?

When PRDM16 expression is low or difficult to detect:

• Signal amplification methods:

  • For IHC/IF: Use tyramide signal amplification (TSA) systems

  • For Western blot: Consider enhanced chemiluminescence substrates or fluorescent secondaries with digital imaging

• Sample enrichment approaches:

  • Use nuclear fractionation to concentrate PRDM16 (as it is a nuclear protein)

  • Consider immunoprecipitation before Western blotting for low abundance samples

  • For brown adipose precursors, cold exposure can increase PRDM16 expression

• Alternative detection strategies:

  • For comprehensive profiling, combine antibody-based detection with mRNA analysis

  • Consider proximity ligation assays (PLA) to detect PRDM16 interactions with known partners

  • For genomic studies, CUT&TAG may provide better sensitivity than traditional ChIP