POLR3D Antibody

What is POLR3D and why is it significant in molecular biology research?

POLR3D (Polymerase RNA III DNA Directed Polypeptide D) is a 44kDa subunit of RNA polymerase III (Pol III), a complex responsible for transcribing small, non-coding RNAs including tRNAs, 5S rRNA, and other short untranslated RNAs. Its significance in molecular biology research stems from its essential role in cellular RNA metabolism. Recent findings have established POLR3D as clinically relevant, with biallelic pathogenic variants in the gene causing RNA polymerase III-related leukodystrophy, a rare hypomyelinating disease . This connection between POLR3D and human disease has intensified research interest in this protein and corresponding antibodies for both basic science and translational medicine purposes.

What applications are POLR3D antibodies most commonly used for?

POLR3D antibodies are most frequently utilized in several key experimental applications:

• Western Blotting (WB): For detection and quantification of POLR3D protein expression levels in cell and tissue lysates

• Enzyme-Linked Immunosorbent Assay (ELISA): For quantitative measurement of POLR3D in solution

• Immunohistochemistry (IHC): For visualizing POLR3D expression patterns in tissue sections

• Immunocytochemistry (ICC): For cellular localization studies

• Chromatin Immunoprecipitation (ChIP): For studying POLR3D binding to chromatin and assessing Pol III occupancy at target genes

The selection of which application to use depends on your specific research question, with WB being the most validated application across commercial antibodies according to the search results.

How do I select the most appropriate POLR3D antibody for my specific research needs?

When selecting a POLR3D antibody, consider these critical factors:

• Epitope recognition: Different antibodies target distinct regions of POLR3D (e.g., AA 281-330, AA 127-156, C-terminus, or middle region) . Choose an epitope that:

  • Is accessible in your experimental conditions

  • Doesn't overlap with functional domains if you're studying protein activity

  • Is not subject to post-translational modifications that might mask recognition

• Host species: Consider compatibility with your experimental design, especially if performing multi-labeling experiments. Rabbit polyclonal antibodies are most common for POLR3D , but mouse monoclonal options are also available .

• Validated applications: Verify that the antibody has been validated for your specific application through published literature or manufacturer data sheets.

• Species reactivity: Confirm cross-reactivity with your model organism. Most POLR3D antibodies react with human and mouse samples , but verification for other species may be necessary.

• Clonality: Polyclonal antibodies offer high sensitivity and recognize multiple epitopes, while monoclonal antibodies provide higher specificity and reproducibility.

What controls should I include when working with POLR3D antibodies?

When designing experiments with POLR3D antibodies, incorporate these essential controls:

• Positive control: Cell lines or tissues known to express POLR3D (most human cell lines express detectable levels)

• Negative control:

  • Primary antibody omission to assess secondary antibody specificity

  • Isotype control (matched immunoglobulin) to evaluate non-specific binding

  • POLR3D-knockdown or knockout samples when available

• Peptide competition assay: Pre-incubation of the antibody with the immunizing peptide should abolish specific signal in most applications.

• Loading controls: For Western blotting, include housekeeping proteins (e.g., β-actin, GAPDH) to normalize POLR3D expression.

• Cross-validation: When possible, use multiple antibodies recognizing different epitopes of POLR3D to confirm observations.

What is the optimal protocol for immunoprecipitation of POLR3D to study its protein interactions?

For effective immunoprecipitation (IP) of POLR3D and its interacting partners:

• Cell lysis buffer optimization:

  • Use a buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% NP-40 or Triton X-100, and protease inhibitors

  • For nuclear proteins like POLR3D, include a nuclear extraction step or use specialized nuclear lysis buffers

  • Consider adding phosphatase inhibitors to preserve phosphorylation states

• Antibody selection:

  • Choose antibodies validated for IP applications

  • Consider epitope accessibility in native conditions

  • FLAG-tagged POLR3D constructs can be used as demonstrated in research where stable clones of POLR3D WT and p.P181S were produced in HeLa cells

• Experimental procedure:

  • Pre-clear lysates with Protein A/G beads to reduce non-specific binding

  • Incubate cleared lysates with 2-5 μg of POLR3D antibody overnight at 4°C

  • Capture complexes with Protein A/G beads for 1-2 hours

  • Wash extensively (4-5 times) with buffer containing reduced detergent concentration

  • Elute with SDS-PAGE loading buffer or under native conditions if preserving complex activity is desired

• Analysis methods:

  • Western blotting to confirm specific interactors

  • Mass spectrometry for unbiased interaction profiling as demonstrated in the study of POLR3D p.P181S missense variant, which showed altered interaction with the PAQosome chaperone complex

How can I optimize Western blotting conditions for detecting POLR3D protein?

For optimal Western blot detection of POLR3D:

• Sample preparation:

  • For nuclear protein POLR3D, use nuclear extraction methods rather than whole-cell lysis

  • Include protease inhibitors to prevent degradation

  • Denature samples at 95°C for 5 minutes in reducing sample buffer

• Gel electrophoresis:

  • Use 10-12% SDS-PAGE gels for optimal resolution of the 44kDa POLR3D protein

  • Load 20-50 μg of total protein per lane depending on expression level

  • Include molecular weight markers spanning 25-75 kDa range

• Transfer conditions:

  • Semi-dry or wet transfer at 100V for 60-90 minutes

  • Use PVDF membrane (0.45 μm) for stronger protein binding

  • Verify transfer efficiency with reversible staining (Ponceau S)

• Blocking and antibody incubation:

  • Block with 5% non-fat milk or BSA in TBST for 1 hour at room temperature

  • Incubate with primary POLR3D antibody (typically 1:500-1:2000 dilution) overnight at 4°C

  • Wash 3-5 times with TBST

  • Incubate with appropriate HRP-conjugated secondary antibody for 1 hour at room temperature

  • Wash extensively before detection

• Detection strategies:

  • Use enhanced chemiluminescence (ECL) for standard detection

  • Consider fluorescent secondary antibodies for quantitative analysis

  • Expected band size: 44kDa, but verify against positive controls

What are the key considerations for using POLR3D antibodies in ChIP-seq experiments?

When performing ChIP-seq with POLR3D antibodies:

• Antibody validation for ChIP:

  • Verify specificity and efficiency in ChIP applications before proceeding to sequencing

  • Test multiple antibodies targeting different epitopes if possible

  • Consider the epitope accessibility in crosslinked chromatin

• Crosslinking optimization:

  • Standard protocol: 1% formaldehyde for 10 minutes at room temperature

  • For transcription factors and associated machinery like POLR3D, optimize crosslinking time (8-15 minutes)

  • Quench with glycine (final concentration 0.125 M)

• Chromatin fragmentation:

  • Sonicate to achieve fragments between 200-500 bp

  • Verify fragmentation efficiency by agarose gel electrophoresis

  • Optimize sonication conditions for your specific cell type

• Immunoprecipitation:

  • Use 3-5 μg of ChIP-grade POLR3D antibody per 25-30 μg of chromatin

  • Include appropriate controls (IgG, input)

  • Consider using a POLR3D antibody that has been validated in previous ChIP studies

• Data analysis considerations:

  • Compare POLR3D binding with other Pol III subunits to confirm complex assembly

  • Analyze enrichment at known Pol III targets (tRNA genes, 5S rRNA)

  • Consider differential binding analysis between experimental conditions

  • As noted in the research, POLR3G- and POLR3GL-containing Pol III complexes may target different Pol III genes, so consider this when interpreting POLR3D ChIP-seq data

What are the common causes of non-specific binding when using POLR3D antibodies?

Non-specific binding is a frequent challenge with POLR3D antibodies. The main causes and solutions include:

• Antibody quality issues:

  • Solution: Use antibodies with >95% purity like those purified by affinity-chromatography using immunogen

  • Solution: Consider testing multiple antibodies from different manufacturers

• Insufficient blocking:

  • Solution: Optimize blocking conditions (5% BSA may be more effective than milk for some applications)

  • Solution: Increase blocking time to 2 hours at room temperature or overnight at 4°C

• Cross-reactivity with related proteins:

  • Solution: Verify specificity using POLR3D knockdown/knockout controls

  • Solution: Use antibodies targeting unique regions of POLR3D that don't share homology with other polymerase subunits

• Excessive antibody concentration:

  • Solution: Perform antibody titration to determine optimal concentration

  • Solution: For Western blots, dilutions between 1:1000-1:5000 are typically effective for commercial POLR3D antibodies

• Denatured vs. native epitopes:

  • Solution: For applications requiring native protein detection, select antibodies validated under non-denaturing conditions

  • Solution: For fixed tissue samples, optimize fixation time to preserve epitope accessibility

How can I resolve inconsistent results between different POLR3D antibodies?

When facing discrepancies between different POLR3D antibodies:

• Epitope mapping analysis:

  • Different antibodies recognize distinct regions of POLR3D (e.g., AA 281-330, AA 127-156, C-terminus)

  • Some epitopes may be masked by protein interactions or post-translational modifications

  • Solution: Map which regions each antibody targets and interpret results accordingly

• Validation with orthogonal methods:

  • Solution: Confirm protein expression using RNA quantification (RT-qPCR)

  • Solution: Use tagged POLR3D constructs as was done in a study using 3x-FLAG-POLR3D WT and p.P181S expression

• Protocol optimization for each antibody:

  • Solution: Adjust incubation times, temperatures, and buffer compositions specifically for each antibody

  • Solution: For Western blots, test multiple blocking agents (milk vs. BSA) and membrane types

• Technical validation:

  • Solution: Verify antibody lot-to-lot consistency with manufacturer

  • Solution: Perform peptide competition assays to confirm specificity of each antibody

• Cross-referencing with literature:

  • Solution: Compare your results with published studies using the same antibodies

  • Solution: Consider the context of experimental conditions in published work

What strategies can address poor signal-to-noise ratio in immunofluorescence with POLR3D antibodies?

To improve signal-to-noise ratio in POLR3D immunofluorescence:

• Fixation optimization:

  • Solution: Compare paraformaldehyde (2-4%) vs. methanol fixation

  • Solution: Optimize fixation time (10-20 minutes) to balance epitope preservation and membrane permeabilization

• Antigen retrieval techniques:

  • Solution: Test heat-induced epitope retrieval (citrate buffer, pH 6.0)

  • Solution: Consider enzymatic retrieval methods if heat-induced methods are ineffective

• Signal amplification methods:

  • Solution: Implement tyramide signal amplification (TSA)

  • Solution: Use biotin-streptavidin amplification systems

  • Solution: Consider secondary antibodies with higher fluorophore conjugation ratios

• Background reduction:

  • Solution: Add 0.1-0.3% Triton X-100 to antibody diluent to reduce non-specific membrane binding

  • Solution: Include 1-5% normal serum from the species of the secondary antibody

  • Solution: Pre-absorb primary antibodies with acetone powder from relevant tissues

• Imaging optimization:

  • Solution: Use confocal microscopy with appropriate pinhole settings

  • Solution: Acquire z-stacks to improve signal detection and specificity

  • Solution: Implement deconvolution algorithms to enhance signal-to-noise ratio

How can POLR3D antibodies be utilized to investigate RNA polymerase III assembly and function?

POLR3D antibodies offer valuable tools for studying Pol III assembly and function:

• Investigating complex assembly:

  • Co-immunoprecipitation with POLR3D antibodies can pull down associated Pol III subunits

  • Analysis by Western blotting or mass spectrometry can reveal complex composition

  • Affinity purification coupled to mass spectrometry (AP-MS) has been used to analyze the POLR3D p.P181S variant, showing normal assembly of Pol III subunits but altered interaction with the PAQosome chaperone complex

• Subcomplex identification:

  • POLR3D is part of a specific Pol III subcomplex

  • Comparing POLR3D pulldown with other subunit pulldowns can reveal assembly intermediates

  • Gel filtration chromatography can separate different POLR3D-containing complexes, as demonstrated for POLR3G and POLR3GL variants

• Functional analysis:

  • ChIP-seq with POLR3D antibodies reveals genome-wide binding patterns of Pol III

  • Compare POLR3D binding with transcriptional output of Pol III targets

  • Developmental or condition-specific changes in POLR3D occupancy can be mapped

• Structure-function relationships:

  • Using antibodies against different POLR3D epitopes can provide insights into accessible regions

  • Combining with crosslinking approaches can map proximity to other complex components

  • Integrating with cryo-EM or X-ray crystallography data for structural interpretation

What role can POLR3D antibodies play in studying pathogenic variants associated with leukodystrophy?

POLR3D antibodies are crucial tools for investigating POLR3D-related leukodystrophy:

• Expression analysis:

  • Western blotting with POLR3D antibodies can assess protein levels in patient samples

  • Immunohistochemistry can reveal tissue-specific expression patterns

  • Comparing mutant and wild-type POLR3D expression can elucidate pathogenic mechanisms

• Functional characterization of variants:

  • Immunoprecipitation can compare interaction partners between wild-type and mutant POLR3D

  • As demonstrated in research, affinity purification coupled to mass spectrometry revealed that the p.P181S missense variant altered Pol III interaction with the PAQosome chaperone complex

  • ChIP assays can assess chromatin occupancy differences between wild-type and mutant POLR3D

• Transcriptional impact assessment:

  • Combining POLR3D antibodies with RNA analysis techniques

  • Studies have shown that biallelic pathogenic variants in POLR3D alter tRNA transcription

  • RT-qPCR assessment revealed decreased expression of POLR3D and other Pol III subunits, as well as decreased expression of Pol III transcripts like 7SK RNA

• Diagnostic applications:

  • POLR3D antibodies might help screen for protein abnormalities in patient samples

  • Immunostaining of patient-derived cells can reveal localization defects

  • POLR3D is the fifth gene encoding Pol III subunits to be associated with a leukodystrophy phenotype

How can POLR3D antibodies be used in conjunction with other techniques to study RNA polymerase III regulation?

Integrating POLR3D antibodies with complementary techniques enhances RNA polymerase III research:

• Combined ChIP-seq and RNA-seq approaches:

  • ChIP-seq with POLR3D antibodies maps genomic occupancy

  • Parallel RNA-seq quantifies transcriptional output

  • Integration reveals relationship between binding and expression

  • This approach was utilized to determine whether POLR3G- and POLR3GL-containing Pol III target different genes

• Proteomics integration:

  • POLR3D immunoprecipitation followed by mass spectrometry identifies interaction partners

  • Crosslinking mass spectrometry (XL-MS) maps protein proximities within complexes

  • Affinity purification coupled to mass spectrometry has been used to analyze POLR3D variants

• Microscopy techniques:

  • Immunofluorescence with POLR3D antibodies visualizes subcellular localization

  • Live-cell imaging with fluorescently tagged POLR3D monitors dynamics

  • Super-resolution microscopy reveals detailed spatial organization of transcription factories

• Functional genomics integration:

  • CRISPR-Cas9 engineering of POLR3D variants combined with antibody-based characterization

  • RNAi-mediated knockdown followed by rescue with mutant constructs

  • tRNA profiling to assess Pol III output, as studies have shown altered tRNA homeostasis in POLR3D-related leukodystrophy

What have recent studies revealed about POLR3D's role in disease pathogenesis?

Recent research has significantly expanded our understanding of POLR3D in disease:

• POLR3D in leukodystrophy:

  • Biallelic pathogenic variants in POLR3D have been identified as a novel genetic cause of POLR3-related leukodystrophy

  • A missense variant (c.541C > T, p.P181S) and an intronic splice site variant (c.656-6G > A) were identified in a patient with clinical and neuroradiological features consistent with POLR3-related leukodystrophy

  • POLR3D is the fifth gene encoding Pol III subunits to be associated with a leukodystrophy phenotype and the sixth gene to be associated with a genetic disease

• Molecular mechanisms:

  • The p.P181S missense variant impacts interaction between Pol III and its chaperone complex

  • Affinity purification coupled to mass spectrometry showed normal assembly of Pol III subunits but altered interaction with the PAQosome chaperone complex

  • The intronic variant is predicted to result in abnormal exon 7 skipping, leading to a premature termination codon (p.V219Gfs*13)

• Transcriptional consequences:

  • Significant decrease in POLR3D RNA-level expression

  • Decreased expression of several other Pol III subunits (POLR3A, POLR3B, POLR1C, POLR3E, and POLR3F)

  • Significant decrease in 7SK RNA and several distinct tRNA genes

  • Altered tRNA homeostasis proposed as a factor in the underlying biology of this hypomyelinating disorder

What emerging techniques might enhance the utility of POLR3D antibodies in research?

Several cutting-edge approaches are enhancing POLR3D antibody applications:

• Proximity labeling techniques:

  • BioID or TurboID fused to POLR3D can identify proximal proteins in living cells

  • APEX2-based proximity labeling allows temporal resolution of interactions

  • These methods complement traditional co-IP with POLR3D antibodies by capturing transient interactions

• Single-cell applications:

  • Single-cell CUT&Tag using POLR3D antibodies can map Pol III occupancy in heterogeneous cell populations

  • Single-cell proteomics may soon enable POLR3D quantification at individual cell resolution

  • Integration with single-cell transcriptomics to correlate POLR3D levels with Pol III transcript abundance

• CRISPR screening applications:

  • CRISPR screens targeting POLR3D regulators combined with antibody-based readouts

  • CRISPR-engineered tagged POLR3D for enhanced antibody detection

  • Base or prime editing to introduce specific POLR3D variants for functional characterization

• Spatial transcriptomics integration:

  • Combining POLR3D immunohistochemistry with spatial transcriptomics

  • Mapping spatial relationships between POLR3D expression and Pol III transcript localization

  • Particularly relevant for studying POLR3D-related leukodystrophy in neural tissues

How might POLR3D antibodies contribute to understanding the distinct functions of RNA polymerase III variants?

POLR3D antibodies can help elucidate functional differences between Pol III variants:

• Distinguishing Pol III complex subtypes:

  • Research has shown that two forms of Pol III exist containing either POLR3G or POLR3GL in separate complexes

  • POLR3D antibodies can help isolate and characterize these distinct complexes

  • Differential co-IP experiments can reveal subunit composition differences

• Genome-wide occupancy mapping:

  • ChIP-seq with POLR3D antibodies can be combined with POLR3G or POLR3GL ChIP-seq

  • This approach has been used to determine whether POLR3G- and POLR3GL-containing Pol III target different Pol III genes

  • Comparison of binding patterns can reveal functional specialization

• Cell type-specific expression:

  • Immunohistochemistry with POLR3D antibodies in different tissues

  • Correlation with POLR3G/POLR3GL expression patterns

  • Understanding developmental or differentiation-associated changes in complex composition

• Disease relevance:

  • POLR3D antibodies can help characterize how different Pol III variants contribute to pathology

  • Investigation of how POLR3D mutations affect assembly with POLR3G versus POLR3GL

  • Potential therapeutic targeting of specific Pol III variants