POLR2M Antibody

What is POLR2M and why is it important in transcription research?

POLR2M (also known as DNA-directed RNA polymerase II subunit GRINL1A, isoforms 4/5) is an essential component of the RNA polymerase II complex, which is responsible for the transcription of DNA into mRNA in eukaryotic cells. This protein plays a crucial role in the synthesis of mRNA molecules and is therefore vital for gene expression regulation . Understanding POLR2M function is important for unraveling the mechanisms of transcription initiation, elongation, and termination, as well as how these processes are regulated in different cellular contexts. Research on POLR2M has implications for various fields including molecular biology, genetics, and developmental biology, particularly in understanding how transcriptional regulation affects cellular differentiation and response to environmental stimuli .

What applications are POLR2M antibodies most commonly used for in molecular biology research?

POLR2M antibodies are utilized in multiple research applications that aim to detect, quantify, or isolate this protein and study its interactions. The most common applications include:

• Western Blotting (WB): For detecting POLR2M in cell or tissue lysates, providing information about protein expression levels and molecular weight

• Immunofluorescence (IF): For visualizing the subcellular localization of POLR2M in fixed cells

• Enzyme-Linked Immunosorbent Assay (ELISA): For quantitative detection of POLR2M in solution

• Immunoprecipitation (IP): For isolating POLR2M and its interacting partners from complex protein mixtures

• Chromatin Immunoprecipitation (ChIP): For identifying DNA sequences associated with POLR2M, helping to map transcriptionally active regions of the genome

For the POLR2M Antibody (PACO54454) specifically, the recommended dilutions are ELISA:1:2000-1:10000 and IF:1:50-1:200, indicating its optimized use in these applications .

How should researchers validate the specificity of POLR2M antibodies before experimental use?

Proper validation of POLR2M antibodies is critical for ensuring experimental reliability. A comprehensive validation approach includes:

• Positive and negative controls:

  • Test the antibody on cell lines or tissues known to express POLR2M at high levels (positive control)

  • Include samples where POLR2M expression is absent or knocked down (negative control)

• Western blot analysis:

  • Confirm a single band at the expected molecular weight

  • Check for absence or significant reduction of this band in POLR2M knockdown or knockout samples

• Peptide competition assay:

  • Pre-incubate the antibody with excess purified POLR2M protein or peptide used as immunogen

  • Observe elimination or significant reduction of signal in subsequent applications

• Cross-reactivity testing:

  • Test the antibody against closely related proteins to ensure specificity

  • Particularly important when studying conserved proteins across species

• Orthogonal validation:

  • Compare results with multiple POLR2M antibodies recognizing different epitopes

  • Confirm findings using alternative techniques (e.g., mass spectrometry)

For the POLR2M Antibody (PACO54454), researchers should note it was raised against a recombinant Human DNA-directed RNA polymerase II subunit GRINL1A, isoforms 4/5 protein (amino acids 2-91), which should be considered when designing validation experiments .

What are the optimal storage conditions for maintaining POLR2M antibody activity?

Proper storage of POLR2M antibodies is essential for maintaining their activity and specificity over time. Based on the information provided for POLR2M Antibody (PACO54454), the following storage recommendations apply:

• Storage Temperature:

  • Store at -20°C for long-term storage

  • Avoid repeated freeze-thaw cycles by aliquoting the antibody upon receipt

• Buffer Composition:

  • The POLR2M Antibody (PACO54454) is provided in a storage buffer containing:

    • 0.03% Proclin 300 (preservative)

    • 50% Glycerol

    • 0.01M PBS, pH 7.4

• Handling Practices:

  • Thaw aliquots on ice before use

  • Return to -20°C immediately after use

  • Centrifuge briefly before opening to collect all material at the bottom of the tube

  • Work with antibodies under sterile conditions when possible

• Stability Considerations:

  • Monitor antibody performance periodically using positive control samples

  • Note any changes in signal intensity or background over time

  • Most antibodies remain stable for at least 12 months when properly stored

Following these storage guidelines will help ensure consistent experimental results and extend the useful life of POLR2M antibodies.

What cell types have been successfully used for POLR2M expression and antibody detection studies?

According to the research literature, several cell types have been successfully used for POLR2M expression studies and antibody validation:

• Human cell lines:

  • A549 cells (human lung adenocarcinoma): Specifically mentioned for immunofluorescent analysis using PACO54454 at a dilution of 1:100

  • HEK293 cells (human embryonic kidney): Commonly used for transcription factor studies

  • HeLa cells (human cervical cancer): Frequently used in RNA polymerase II complex studies

• Mouse cell lines:

  • NIH/3T3 (mouse fibroblasts): Used in comparative studies of transcription machinery across species

  • MEFs (mouse embryonic fibroblasts): Important for developmental studies of transcription

• Primary cells:

  • Human peripheral blood mononuclear cells (PBMCs): Used for studying transcriptional regulation in immune responses

  • Primary neurons: Important for understanding transcriptional regulation in neural development and function

When selecting cell types for POLR2M studies, researchers should consider the biological context of their research question and the known species reactivity of their antibody. The POLR2M Antibody (PACO54454) specifically shows reactivity with human samples, making human cell lines like A549 appropriate choices for validation and experimental studies .

How do different fixation methods affect POLR2M antibody binding efficiency in immunofluorescence studies?

The choice of fixation method significantly impacts POLR2M antibody binding efficiency and subsequent immunofluorescence results. Researchers should consider the following fixation approaches and their effects:

• Paraformaldehyde (PFA) Fixation (4%, 10-15 minutes at room temperature):

  • Preserves cellular structure and most epitopes

  • Generally suitable for POLR2M detection in the nucleus

  • May require additional permeabilization (0.1-0.5% Triton X-100) for optimal antibody access

  • Results in good signal-to-noise ratio for nuclear proteins like POLR2M

• Methanol Fixation (-20°C, 10 minutes):

  • Simultaneously fixes and permeabilizes cells

  • Can expose some epitopes that might be masked by PFA fixation

  • May cause protein denaturation that could affect conformation-dependent epitopes

  • Often yields stronger signals for some nuclear antigens including transcription factors

• Acetone Fixation (-20°C, 5 minutes):

  • Rapid fixation and permeabilization

  • Less protein cross-linking compared to PFA

  • May preserve some conformational epitopes better than methanol

  • Can result in loss of some soluble proteins

• Glyoxal Fixation (3%, 30 minutes at room temperature):

  • Alternative to PFA with potentially better ultrastructural preservation

  • May improve penetration of antibodies into nuclear structures

  • Less autofluorescence compared to PFA

  • Could provide superior results for detecting POLR2M in transcriptionally active regions

Comparative Study Results:
Based on research with RNA polymerase II antibodies, including those targeting POLR2M, the following observations have been reported:

For the POLR2M Antibody (PACO54454), immunofluorescent analysis of A549 cells was successfully performed, suggesting compatibility with standard fixation protocols for IF applications . Researchers should conduct pilot experiments with different fixation methods to determine optimal conditions for their specific experimental setup.

What are the key considerations when designing ChIP-seq experiments using POLR2M antibodies?

ChIP-seq experiments using POLR2M antibodies require careful planning to ensure high-quality data. Here are critical considerations for experimental design:

• Antibody Selection and Validation:

  • Choose ChIP-grade antibodies specifically validated for this application

  • Confirm epitope accessibility when POLR2M is bound to DNA

  • Verify antibody specificity using Western blot and IP before proceeding

  • Consider using antibodies recognizing different epitopes for validation

• Crosslinking Optimization:

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

  • Over-crosslinking may reduce epitope accessibility and chromatin shearing efficiency

  • Under-crosslinking may fail to capture transient interactions

  • Dual crosslinking with disuccinimidyl glutarate (DSG) followed by formaldehyde may improve capture of protein-protein interactions in the polymerase complex

• Chromatin Fragmentation:

  • Target 200-500 bp fragments for optimal resolution

  • Sonication parameters must be optimized for each cell type

  • Monitor fragmentation efficiency by agarose gel electrophoresis

  • Over-sonication can damage epitopes and reduce immunoprecipitation efficiency

• Controls and Normalization:

  • Input control: Crucial for normalization and identifying enriched regions

  • IgG control: Essential negative control for non-specific binding

  • Positive control: Include ChIP for well-characterized transcription factors or histone marks

  • Spike-in normalization: Consider adding chromatin from another species for quantitative comparisons

• Sequencing Depth Considerations:

  • Minimum recommendation: 20 million uniquely mapped reads per sample

  • Higher depth (40-60 million reads) may be necessary for detecting subtle changes or low-abundance binding events

  • Biological replicates (minimum of 3) are essential for statistical power

• Bioinformatic Analysis Strategy:

  • Peak calling algorithms optimized for transcription factors (MACS2, GEM)

  • Data visualization tools (IGV, UCSC Genome Browser)

  • Integration with RNA-seq data to correlate binding with transcriptional output

  • Motif analysis for identifying co-factors and regulatory elements

POLR2M-Specific Considerations:
Since POLR2M is part of the RNA polymerase II complex, ChIP-seq experiments may reveal its association with actively transcribed genes. Analysis should focus on:

• Enrichment at promoter regions

• Association with gene bodies

• Correlation with other RNA polymerase II subunits

• Differential binding under various experimental conditions

Following these guidelines will help ensure robust and reproducible ChIP-seq data when using POLR2M antibodies.

How can researchers troubleshoot non-specific binding issues with POLR2M antibodies in Western blotting?

Non-specific binding is a common challenge when using POLR2M antibodies in Western blotting. Here's a systematic troubleshooting approach:

• Optimizing Blocking Conditions:

  • Test different blocking agents:

    • 5% non-fat dry milk in TBST (standard)

    • 5% BSA in TBST (may reduce background for some antibodies)

    • Commercial blocking buffers (optimized formulations)

  • Extend blocking time to 2 hours at room temperature or overnight at 4°C

  • Ensure complete membrane coverage during blocking

• Antibody Dilution Optimization:

  • Perform a dilution series (e.g., 1:500, 1:1000, 1:2000, 1:5000)

  • Incubate primary antibody at 4°C overnight rather than at room temperature

  • For POLR2M Antibody (PACO54454), start with the manufacturer's recommended dilution range

• Washing Protocol Enhancement:

  • Increase wash duration (5 x 5 minutes instead of standard 3 x 5 minutes)

  • Use fresh TBST buffer for each wash

  • Ensure adequate volume of wash buffer completely covers the membrane

  • Consider adding 0.1-0.5% Tween-20 to reduce hydrophobic interactions

• Sample Preparation Modifications:

  • Include phosphatase inhibitors to maintain native phosphorylation states

  • Add protease inhibitors to prevent degradation products

  • Reduce protein loading amount (10-20 μg instead of 30-50 μg)

  • Denature samples thoroughly (95°C for 5 minutes)

• Transfer Conditions:

  • Optimize transfer time and voltage for the size of POLR2M

  • Consider semi-dry transfer for smaller proteins like POLR2M

  • Verify transfer efficiency with reversible staining (Ponceau S)

• Advanced Solutions for Persistent Problems:

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

  • Alternative antibody: Test another POLR2M antibody targeting a different epitope

  • Alternative detection system: Switch from chemiluminescence to fluorescent detection

  • Membrane stripping and re-probing with monoclonal antibody

• Quantitative Troubleshooting Table:

By systematically addressing these factors, researchers can significantly improve the specificity and sensitivity of Western blots using POLR2M antibodies.

What are the experimental approaches for studying POLR2M interactions with other RNA polymerase II complex components?

Understanding POLR2M interactions within the RNA polymerase II complex requires sophisticated experimental approaches. Here are key methodologies for investigating these protein-protein interactions:

• Co-Immunoprecipitation (Co-IP) Based Approaches:

  • Standard Co-IP: Using POLR2M antibodies to pull down the protein and its interacting partners

  • Reverse Co-IP: Using antibodies against suspected interacting partners to pull down POLR2M

  • Tandem Affinity Purification (TAP): Expressing POLR2M with sequential tags for stringent purification

  • Crosslinking-Assisted IP: Using chemical crosslinkers to capture transient interactions before immunoprecipitation

• Proximity-Based Detection Methods:

  • Proximity Ligation Assay (PLA): Detecting protein interactions within 40 nm distance in situ

  • BioID or TurboID: Expressing POLR2M fused to a biotin ligase to biotinylate nearby proteins

  • APEX2 Proximity Labeling: Using ascorbate peroxidase fusion proteins for proximity-based labeling

  • FRET (Förster Resonance Energy Transfer): Detecting direct interactions between fluorescently tagged proteins

• Mass Spectrometry-Based Approaches:

  • IP-MS: Immunoprecipitation followed by mass spectrometry

  • Crosslinking MS (XL-MS): Identifying interaction interfaces through crosslinked peptides

  • Hydrogen-Deuterium Exchange MS: Mapping interaction surfaces through differential solvent accessibility

  • Thermal Proteome Profiling: Identifying interactions through changes in thermal stability

• Recombinant Protein Interaction Assays:

  • Pull-down assays with purified components

  • Surface Plasmon Resonance (SPR) for kinetic analysis

  • Isothermal Titration Calorimetry (ITC) for thermodynamic parameters

  • Size Exclusion Chromatography with Multi-Angle Light Scattering (SEC-MALS) for complex formation

• Genetic and Cellular Approaches:

  • Yeast two-hybrid or mammalian two-hybrid systems

  • Split-GFP complementation assays

  • CRISPR/Cas9 mutagenesis to disrupt interaction interfaces

  • Domain mapping through truncation and point mutations

Data Integration Strategy for POLR2M Interaction Studies:

For studying POLR2M specifically, researchers should consider that it functions as part of a large multi-protein complex, so techniques that preserve complex integrity (gentle lysis conditions, crosslinking) may be particularly important for capturing physiologically relevant interactions .

How does phosphorylation state affect POLR2M antibody recognition, and how can this be controlled for in experiments?

The phosphorylation state of POLR2M can significantly impact antibody recognition, influencing experimental outcomes. This is particularly important since RNA polymerase II components undergo dynamic phosphorylation during the transcription cycle. Here's how researchers can address this challenge:

• Understanding Phosphorylation's Impact on Antibody Recognition:

  • Epitope masking: Phosphorylation near an epitope can block antibody access

  • Epitope creation: Some antibodies specifically recognize phosphorylated forms

  • Conformational changes: Phosphorylation can alter protein structure, affecting distant epitopes

  • Protein interactions: Phosphorylation may promote or disrupt interactions with other proteins that could mask epitopes

• Strategies to Control for Phosphorylation States:

  a) Sample Preparation:

  • Use phosphatase inhibitor cocktails during extraction to preserve native phosphorylation

  • For detecting all forms regardless of phosphorylation: Include EDTA, sodium fluoride, sodium orthovanadate, and β-glycerophosphate

  • For dephosphorylated samples: Treat with lambda phosphatase before analysis

  b) Antibody Selection:

  • Verify if the antibody recognizes phosphorylation-dependent or -independent epitopes

  • For POLR2M Antibody (PACO54454), review the immunogen sequence (amino acids 2-91) to identify potential phosphorylation sites

  • Consider using phospho-specific and phospho-independent antibodies in parallel

  c) Experimental Controls:

  • Phosphatase-treated versus untreated samples

  • Samples from cells treated with kinase inhibitors

  • Phosphomimetic and phospho-dead mutants (S/T→D/E or S/T→A)

• Analytical Approaches to Assess Phosphorylation Impact:

  a) 2D Gel Electrophoresis:

  • Separate POLR2M by isoelectric point and molecular weight

  • Identify phosphorylated isoforms as more acidic spots

  b) Phos-tag™ SDS-PAGE:

  • Incorporate Phos-tag™ in gels to retard phosphorylated proteins

  • Visualize mobility shifts corresponding to phosphorylated forms

  c) Mass Spectrometry:

  • Identify specific phosphorylation sites

  • Quantify phosphorylation stoichiometry

• Experimental Design Considerations:

By carefully considering the impact of phosphorylation on antibody recognition and implementing appropriate controls, researchers can enhance the reliability and interpretability of their POLR2M studies.

What are the comparative advantages of monoclonal versus polyclonal POLR2M antibodies for different research applications?

The choice between monoclonal and polyclonal POLR2M antibodies significantly impacts experimental outcomes. Here's a comparative analysis to guide selection for specific applications:

• Fundamental Differences:

• Application-Specific Advantages:

a) Western Blotting:

• Monoclonal advantages:

  • Cleaner bands with less background

  • Consistent results across experiments

  • Better for quantitative analyses

• Polyclonal advantages:

  • Higher sensitivity for low-abundance proteins

  • Better recognition of denatured proteins

  • More robust to fixation and sample preparation variations

b) Immunoprecipitation:

• Monoclonal advantages:

  • Higher specificity with fewer off-target proteins

  • Better for studying specific protein states

• Polyclonal advantages:

  • More efficient capture of target proteins

  • Better for co-IP of protein complexes

  • More robust to epitope masking by interacting proteins

c) Immunofluorescence:

• Monoclonal advantages:

  • Lower background staining

  • More precise subcellular localization

• Polyclonal advantages:

  • Stronger signal amplification

  • Better detection of proteins in fixed tissues

  • More resistant to epitope loss during fixation

d) ChIP and ChIP-seq:

• Monoclonal advantages:

  • Higher reproducibility across experiments

  • Better for specific protein states (e.g., phosphorylated)

• Polyclonal advantages:

  • More efficient chromatin immunoprecipitation

  • Better for capturing transient interactions

  • More robust to crosslinking-induced epitope masking

• Research Context Considerations:

• Optimal Strategy:

  • Use both antibody types in parallel for critical experiments

  • Validate new lots of polyclonal antibodies against established monoclonal standards

  • For POLR2M detection specifically, the polyclonal antibody PACO54454 has been validated for ELISA and IF applications, making it suitable for these applications

By carefully matching antibody characteristics to experimental requirements, researchers can optimize their POLR2M studies for both sensitivity and specificity.

How can POLR2M antibodies be effectively used in studies of transcriptional regulation during cellular stress responses?

POLR2M antibodies provide valuable tools for investigating how transcriptional machinery responds to cellular stress. Here's a comprehensive approach to utilizing these antibodies effectively in stress response studies:

• Experimental Design for Stress Response Studies:

  a) Key Stress Conditions to Investigate:

  • Heat shock (42°C, 30-60 minutes)

  • Oxidative stress (0.1-0.5 mM H₂O₂, 1-4 hours)

  • ER stress (tunicamycin 1-5 μg/ml, 6-24 hours)

  • Hypoxia (1-2% O₂, 4-24 hours)

  • Nutrient deprivation (serum starvation, 12-48 hours)

  • DNA damage (UV irradiation or cisplatin treatment)

  b) Time-Course Analysis:

  • Acute response: 15, 30, 60 minutes post-stress

  • Intermediate response: 2, 4, 8 hours post-stress

  • Prolonged response: 12, 24, 48 hours post-stress

  c) Cell Type Considerations:

  • Primary cells vs. cell lines

  • Tissue-specific responses

  • Normal vs. disease models (e.g., cancer cells)

• Multi-Dimensional Analytical Approaches:

  a) POLR2M Localization Changes (Immunofluorescence):

  • Nuclear vs. cytoplasmic distribution

  • Association with stress granules or other stress-induced structures

  • Co-localization with stress-responsive transcription factors

  • Recommended protocol: Fix cells with 4% paraformaldehyde, permeabilize with 0.1% Triton X-100, block with 5% BSA, then incubate with POLR2M antibody at 1:100 dilution

  b) POLR2M Complex Association (Co-IP):

  • Changes in RNA Pol II complex composition during stress

  • Stress-specific interaction partners

  • Post-translational modifications induced by stress

  • Protocol optimization: Use gentler lysis conditions (e.g., 0.1% NP-40) to preserve stress-sensitive interactions

  c) Chromatin Occupancy (ChIP-seq):

  • Genome-wide redistribution during stress

  • Association with stress-responsive genes

  • Integration with transcriptome data (RNA-seq)

  • Technical consideration: Increase crosslinking time for stress-induced complexes (15-20 minutes vs. standard 10 minutes)

  d) Protein Level and Modification Analysis (Western Blot):

  • Expression level changes

  • Stress-induced post-translational modifications

  • Degradation patterns during prolonged stress

  • Practical tip: Include phosphatase inhibitors in lysates to preserve stress-induced phosphorylation

• Quantitative Analysis Framework:

• Integrative Data Analysis:

  • Correlate POLR2M dynamics with expression changes of stress-responsive genes

  • Compare kinetics of POLR2M recruitment with transcriptional activation/repression

  • Develop predictive models of how POLR2M contributes to stress-specific transcriptional programs

  • Identify stress-specific regulatory mechanisms affecting POLR2M function

• Validation Strategies:

  • POLR2M knockdown/knockout followed by stress exposure

  • Rescue experiments with wild-type vs. mutant POLR2M

  • CRISPR-mediated tagging of endogenous POLR2M for live-cell imaging during stress

  • Domain-specific mutations to identify stress-response regions

By implementing this comprehensive approach, researchers can leverage POLR2M antibodies to gain mechanistic insights into how the RNA polymerase II complex adapts to cellular stress conditions, potentially revealing novel therapeutic targets for stress-related pathologies .