POLR2F Antibody

What is POLR2F and why is it important in transcription research?

POLR2F (RNA polymerase II subunit F) is a 14 kDa subunit of RNA polymerase II that plays a critical role in the transcription process. It serves as a key component of the RNA polymerase II complex responsible for transcribing protein-coding genes . This protein is highly conserved across species (human, mouse, rat) and is essential for understanding fundamental mechanisms of gene expression and regulation.

For researchers targeting this protein:

• POLR2F's involvement in transcription makes it valuable for studying gene expression dysregulation

• The protein is part of larger complexes including the core PAF complex (RNA polymerase-associated factor)

• POLR2F has been found to associate with other proteins involved in RNA pol II pausing/restarting and elongation

How do I select the appropriate POLR2F antibody for my specific application?

Selection should be based on:

• Application compatibility: Different antibodies are optimized for specific applications with varying recommended dilutions:

• Species reactivity: Confirm reactivity with your target species (human, mouse, rat)

• Clonality: Choose between:

  • Polyclonal antibodies: Broader epitope recognition (e.g., product 15334-1-AP)

  • Monoclonal antibodies: Higher specificity (e.g., product 68511-1-Ig)

• Validation data: Review published validation data for the specific antibody

How can I optimize Western blot protocols for POLR2F detection?

For optimal Western blot results with POLR2F antibodies:

• Sample preparation:

  • Use appropriate lysis buffers containing protease inhibitors

  • For nuclear proteins like POLR2F, nuclear extraction protocols may improve results

• Loading controls:

  • Use housekeeping proteins of different molecular weights from POLR2F (14 kDa)

  • Consider nuclear-specific loading controls when appropriate

• Protocol optimization:

  • Begin with manufacturer's recommended dilution (typically 1:500-1:16000)

  • Observed molecular weight should be approximately 14-17 kDa, though some antibodies may detect at 23 kDa

  • Extended blocking times (1-2 hours) may reduce background

  • Optimize primary antibody incubation (4°C overnight often yields best results)

• Troubleshooting:

  • If high background occurs, increase dilution factor

  • If weak signal occurs, reduce dilution or extend exposure time

  • Consider enhanced chemiluminescence substrates for improved sensitivity

What controls should be included when performing immunoprecipitation with POLR2F antibodies?

Proper controls for POLR2F immunoprecipitation experiments include:

• Input control: 5-10% of starting material before immunoprecipitation

• Negative controls:

  • IgG control: Use same species IgG at equivalent concentration to POLR2F antibody

  • No-antibody control: Perform IP procedure without primary antibody

• Positive controls:

  • IP with antibodies against known POLR2F-interacting proteins (e.g., other RNA polymerase II subunits)

  • Use of tagged-POLR2F constructs with corresponding tag antibodies

• Validation approaches:

  • Reciprocal IP (pull-down with antibody against interacting partner)

  • Mass spectrometry analysis of IP products

Research has shown POLR2F co-immunoprecipitates with components of the PAF complex, FACT complex, SUPT5H, SUPT6H, and DNA topoisomerase I, indicating its role in transcription elongation complexes .

How can POLR2F antibodies be effectively used in ChIP and ChIP-seq experiments?

For chromatin immunoprecipitation applications:

• Crosslinking optimization:

  • Standard formaldehyde fixation (1%) for 10 minutes at room temperature

  • Alternative dual crosslinking with DSG followed by formaldehyde may improve results for transcription factors

• Sonication parameters:

  • Optimize sonication to achieve 200-500 bp DNA fragments

  • Verify fragmentation using agarose gel electrophoresis

• ChIP protocol specifics:

  • Use 3-5 μg of POLR2F antibody per IP reaction

  • Include input controls (5-10% of starting chromatin)

  • Include IgG control from same species as POLR2F antibody

• Data analysis considerations:

  • POLR2F binding may correlate with active transcription regions

  • Compare POLR2F occupancy with other Pol II subunits

  • Analyze with reference to transcription start sites and gene bodies

One study showed that RNA pol II elongation factors including POLR2F can be detected by ChIP-seq at actively transcribed genes, providing insights into the mechanism of AID activity at Ig loci .

How do phosphorylation states affect POLR2F antibody recognition and what methodologies can address this challenge?

Phosphorylation of RNA polymerase II subunits significantly impacts antibody recognition:

• Impact of phosphorylation:

  • RNA Pol II exists in hypophosphorylated (IIa) and hyperphosphorylated (IIo) forms

  • Phosphorylation states change during transcription and can mask epitopes

  • Phosphorylation patterns may be conserved between yeast and humans

• Methodological approaches:

  • Use phosphorylation-independent POLR2F antibodies for total POLR2F detection

  • Employ phosphatase treatment of samples to eliminate phosphorylation-dependent recognition issues

  • For phosphorylation research, utilize specialized antibodies targeting particular phosphorylation states

• Advanced analysis:

  • Combine IP with mass spectrometry to identify phosphorylation sites

  • Use phospho-specific antibodies in sequential IPs to identify subpopulations

Research has demonstrated that "gross changes in CTD phosphorylation patterns during transcription may be more conserved in yeast and humans than recognized previously" , which may affect antibody recognition of POLR2F in different transcriptional states.

What strategies can be employed to study POLR2F interactions within transcription complexes?

To investigate POLR2F within larger complexes:

• Co-immunoprecipitation approaches:

  • Use native conditions to preserve protein-protein interactions

  • Sequential IPs to isolate specific subcomplexes

  • Cross-linking followed by IP to capture transient interactions

• Size exclusion chromatography combined with immunoblotting:

  • Hyperphosphorylated RNA Pol II can be isolated in specific fractions

  • Follow with immunoblotting using POLR2F antibodies to track complex formation

• Proximity labeling techniques:

  • BioID or APEX2 fusion with POLR2F to identify proximal proteins

  • Mass spectrometry analysis of labeled proteins

• Advanced microscopy:

  • Immunofluorescence co-localization with other polymerase subunits

  • Super-resolution microscopy for detailed spatial relationships

Studies have identified POLR2F associations with multiple complexes involved in transcription regulation, including "the core PAF complex (RNA polymerase-associated factor; PAF1, CTR9, LEO1), FACT complex (SSRP1, SUPT16H), SUPT5H, SUPT6H, and DNA topo I" .

How can I validate the specificity of POLR2F antibodies in my experimental system?

Multiple validation approaches should be employed:

• Molecular techniques:

  • siRNA/shRNA knockdown of POLR2F followed by Western blot analysis

  • CRISPR/Cas9 knockout or knockdown validation when possible

  • Overexpression of tagged POLR2F as positive control

• Cross-validation:

  • Compare staining patterns between multiple antibodies targeting different POLR2F epitopes

  • Compare observed molecular weight with predicted (14 kDa for POLR2F)

• Peptide competition assays:

  • Pre-incubate antibody with immunizing peptide before application

  • Should eliminate specific staining if antibody is specific

• Multi-application validation:

  • Confirm consistent results across different techniques (WB, IF, IHC)

  • Multiple cell lines or tissues for broader validation

Several POLR2F antibodies have been validated through extensive testing in cell lines (HeLa, NIH/3T3, C6, A431, MCF-7) and tissues (human cervical cancer, human placenta) .

Why do I observe discrepancies in molecular weight detection between different POLR2F antibodies?

Molecular weight variations may occur due to:

• Technical factors:

  • Different gel systems and running conditions affect apparent molecular weight

  • Various sample preparation methods can influence protein migration

  • Post-translational modifications alter migration patterns

• Antibody-specific factors:

  • Some antibodies (e.g., 15334-1-AP) detect POLR2F at 14 kDa

  • Others (e.g., 68511-1-Ig) observe it at 23 kDa

  • Antibody epitope location may affect detection of modified forms

• Biological factors:

  • POLR2F may exist in different isoforms

  • Post-translational modifications (particularly phosphorylation)

  • Protein complexes that resist complete denaturation

• Resolution approaches:

  • Run positive control samples alongside experimental samples

  • Test multiple antibodies targeting different epitopes

  • Perform peptide competition assay to confirm specific bands

How can POLR2F antibodies contribute to understanding transcriptional regulation in disease models?

POLR2F antibodies enable multiple research approaches:

• Cancer research applications:

  • Compare POLR2F occupancy at oncogenes in normal vs. cancer cells

  • Study transcriptional dysregulation through POLR2F complex alterations

  • Examine nuclear localization changes in cancer progression

• Immune system research:

  • POLR2F is involved in the PAF complex which plays a role in antibody diversification

  • Knockdown experiments in murine B cells have demonstrated the biological relevance of the PAF complex in class switch recombination

  • POLR2F antibodies can help track RNA polymerase II components during immune responses

• Neurodegenerative disease studies:

  • Investigate transcriptional dysregulation in neuronal cells

  • Compare RNA polymerase II complex integrity across disease models

  • Track POLR2F localization in stress conditions

• Methodology considerations:

  • Combine POLR2F ChIP-seq with RNA-seq for comprehensive analysis

  • Use inducible disease models to track temporal changes

  • Consider single-cell approaches for heterogeneous samples

Research has shown that "RNA pol II elongation factors associate with AID on chromatin," providing "insight into the mechanism of AID activity at Ig loci" , which has implications for understanding immune disorders.

What considerations are important when using POLR2F antibodies in multi-parameter flow cytometry or CyTOF experiments?

For flow cytometry applications with POLR2F antibodies:

• Sample preparation protocol:

  • Nuclear permeabilization is essential (POLR2F is nuclear)

  • Test different fixation and permeabilization reagents

  • Optimize incubation times to ensure antibody penetration

• Antibody selection factors:

  • Choose antibodies that work in flow cytometry applications

  • Confirm epitope accessibility in fixed/permeabilized cells

  • Select appropriately conjugated antibodies for panel design

• Panel design considerations:

  • Pair POLR2F antibody with markers for cell cycle phases

  • Include markers for specific cell types of interest

  • Consider transcription-associated markers for correlation

• Controls and validation:

  • Include FMO (fluorescence minus one) controls

  • Validate staining pattern by imaging flow cytometry

  • Confirm results with alternative methods (Western blot, microscopy)

• Data analysis considerations:

  • Account for autofluorescence from fixed cells

  • Use appropriate gating strategies for nuclear proteins

  • Consider cell cycle effects on POLR2F expression