POLR2H Antibody

What is POLR2H and why is it important in research?

POLR2H, also known as hRPB8, is a 150 amino acid protein that localizes in the nucleolus. It functions as a subunit of RNA polymerase II and is part of the DNA-directed RNA polymerases I, II, and III complex . The protein has a molecular weight of approximately 17 kDa and is encoded by the POLR2H gene (Gene ID: 5437) . POLR2H plays a critical role in transcription processes, making it an important research target for studies involving gene expression, transcriptional regulation, and associated diseases. The protein is highly expressed across multiple tissue types, which explains its importance as a research target for understanding basic cellular mechanisms . Antibodies against POLR2H are valuable tools for investigating its expression patterns, interactions with other proteins, and role in various cellular processes.

Based on current research, commercially available POLR2H antibodies show reactivity with samples from multiple species:

The high conservation of POLR2H across mammalian species contributes to this cross-reactivity pattern. When conducting experiments with samples from species not listed above, preliminary validation tests are strongly recommended to ensure antibody specificity and binding efficiency .

How should I optimize POLR2H antibody dilutions for different experimental applications?

Optimizing POLR2H antibody dilutions is essential for balancing specific signal detection with minimal background. Begin with the manufacturer's recommended dilution ranges and adjust based on your specific experimental conditions:

For Western blotting:

• Start with a mid-range dilution (1:500) and adjust based on signal intensity

• For high abundance targets, use higher dilutions (1:1000-1:2000)

• For low abundance targets, use lower dilutions (1:200-1:500)

• Run a gradient of at least three different dilutions to determine optimal concentration

• Include a loading control antibody to normalize expression levels

For immunohistochemistry:

• Begin with a 1:100 dilution and adjust based on signal-to-noise ratio

• Perform antigen retrieval with TE buffer (pH 9.0) or alternatively with citrate buffer (pH 6.0)

• Include positive control tissues (human breast cancer tissue has been validated)

• Test multiple dilutions on serial sections to identify optimal conditions

For immunofluorescence:

• Start with a 1:100 dilution for cultured cells (HeLa cells have been validated)

• Include appropriate counterstains (DAPI for nuclei)

• Test specificity with blocking peptides if available

For immunoprecipitation:

• Use 0.5-4.0 μg antibody for 1.0-3.0 mg of total protein lysate

• Consider pre-clearing lysates to reduce non-specific binding

• Include negative controls (isotype control or pre-immune serum)

Remember that these recommendations serve as starting points, and the optimal dilution may vary depending on sample type, fixation method, and detection system employed .

What are the recommended storage conditions for maximizing POLR2H antibody stability and performance?

Proper storage of POLR2H antibodies is critical for maintaining their performance characteristics over time. Based on manufacturer recommendations:

For long-term storage:

• Store at -20°C in small aliquots to minimize freeze-thaw cycles

• The antibody is stable for up to one year from the date of receipt when properly stored

• Some formulations (83439-1-PBS) should be stored at -80°C and shipped with ice packs

For buffer composition:

• Most POLR2H antibodies are supplied in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3

• Some formulations are available in PBS only (BSA and azide free) for applications requiring conjugation

For handling:

• Avoid repeated freeze-thaw cycles as they can lead to denaturation and decreased activity

• Allow the antibody to equilibrate to room temperature before opening

• When removing an aliquot, immediately return the stock to -20°C or -80°C

• Centrifuge the vial briefly before opening to ensure all liquid is at the bottom

For small volume antibodies (20μl sizes):

• These may contain 0.1% BSA as a stabilizer

• Aliquoting is unnecessary for -20°C storage for these formats

Following these storage guidelines will help maintain antibody performance and extend shelf life for reliable experimental results .

How do I validate POLR2H antibody specificity for my experimental system?

Validating antibody specificity is a crucial step before embarking on research with POLR2H antibodies. A comprehensive validation approach should include:

• Molecular weight verification:

  • Confirm that the observed molecular weight in Western blots matches the expected 17 kDa size of POLR2H

  • Look for single, clean bands rather than multiple non-specific bands

• Positive and negative controls:

  • Use cell lines with known POLR2H expression (validated examples include HL-60, HeLa, and MCF-7 cells)

  • Include tissue samples with confirmed expression (mouse kidney, mouse/rat liver)

  • Consider POLR2H knockdown or knockout samples as negative controls

• Cross-validation with multiple antibodies:

  • Compare results from different antibody clones targeting different epitopes of POLR2H

  • Consistency between antibodies increases confidence in specificity

• Peptide competition assay:

  • Pre-incubate the antibody with excess immunizing peptide before application

  • Specific signals should be abolished or significantly reduced

• Immunoprecipitation validation:

  • Confirm that the antibody can immunoprecipitate POLR2H from complex samples

  • Verify the identity of the precipitated protein by mass spectrometry when possible

• Cross-species reactivity assessment:

  • If working with non-human samples, verify reactivity in your specific species

  • Compare sequence homology between the immunogen and your species of interest

Remember that antibody validation is not a one-time process but should be repeated periodically, especially when changing experimental conditions or starting new batches of antibody .

How do I troubleshoot inconsistent results with POLR2H antibody across different cell lines?

Inconsistent POLR2H antibody performance across cell lines can result from multiple factors. A systematic troubleshooting approach includes:

Biological variables:

• Expression level variations: POLR2H expression may vary substantially between cell types despite being widely expressed

• Post-translational modifications: Different cell lines may exhibit varying patterns of modifications affecting epitope accessibility

• Protein interactions: Cell-specific protein complexes may mask epitopes in certain contexts

Technical considerations:

• Lysis buffer optimization: Test multiple lysis buffers with different detergent concentrations

  • RIPA buffer for most applications

  • NP-40 buffer for preserving protein-protein interactions

  • SDS buffer for more stringent extraction

• Protocol adjustments for cell type:

  • Adherent cells (like HeLa) may require different harvesting methods than suspension cells (like HL-60)

  • Primary cells often need gentler lysis conditions than immortalized lines

• Blocking optimization:

  • Test BSA vs. non-fat dry milk as blocking agents

  • Consider cell-line specific background by including pre-immune serum controls

Validation strategies:

• Create a dilution series of positive control lysate (HeLa or MCF-7) alongside test samples

• Run standardized amounts of recombinant POLR2H as reference points

• Include multiple positive controls from different tissue/cell origins

When encountering resistance to optimization, consider specialized approaches:

• Immunoprecipitation followed by Western blotting to enrich for POLR2H

• Alternative antibodies targeting different epitopes of the protein

• mRNA expression analysis (qPCR) to correlate with protein detection results

Document all optimization steps systematically, as cell line-specific protocols may need to be established for consistent results .

What are the considerations for using POLR2H antibody in co-immunoprecipitation experiments?

When designing co-immunoprecipitation (co-IP) experiments to study POLR2H protein interactions, several critical considerations should be addressed:

Antibody selection:

• Choose antibodies validated specifically for immunoprecipitation

• For POLR2H, use 0.5-4.0 μg antibody for 1.0-3.0 mg of total protein lysate

• Consider the epitope location to avoid interfering with protein-protein interaction sites

Lysis conditions:

• Use non-denaturing lysis buffers to preserve protein-protein interactions

• Recommended buffer: 150 mM NaCl, 1.0% NP-40 or Triton X-100, 50 mM Tris pH 8.0

• Include protease and phosphatase inhibitors to prevent degradation

• Optimize detergent concentration to balance extraction efficiency with preservation of interactions

Control experiments:

• IgG control: Include species-matched IgG to identify non-specific binding

• Reverse co-IP: Confirm interactions by immunoprecipitating with antibodies against the interacting partner

• Input control: Load 5-10% of pre-cleared lysate to verify protein presence

Experimental conditions:

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

• Incubate antibody with lysate overnight at 4°C for optimal binding

• For detecting transient interactions, consider crosslinking approaches

Detection strategies:

• Western blot using specific antibodies against expected interacting partners

• Mass spectrometry for unbiased identification of novel interaction partners

Particular considerations for POLR2H co-IP:

• POLR2H functions as part of large RNA polymerase complexes, so gentle lysis conditions are critical

• Mouse liver tissue has been validated for POLR2H immunoprecipitation

• Due to its role in transcription complexes, DNase/RNase treatment may help distinguish direct protein interactions from DNA/RNA-mediated associations

By carefully addressing these considerations, researchers can effectively use POLR2H antibodies to identify and characterize its interaction partners in various biological contexts .

How do polyclonal and monoclonal POLR2H antibodies compare in specificity and application performance?

The choice between polyclonal and monoclonal POLR2H antibodies significantly impacts experimental outcomes based on their inherent characteristics and performance in different applications:

Application-specific considerations:

For Western blotting:

• Polyclonal antibodies (15086-1-AP, STJ29028) provide good sensitivity at 1:200-1:2000 dilutions

• Monoclonal/recombinant antibodies may offer cleaner backgrounds for quantitative analysis

For immunohistochemistry:

• Polyclonal antibodies have been validated for IHC at 1:20-1:200 dilutions

• Consider antigen retrieval requirements (TE buffer pH 9.0 or citrate buffer pH 6.0)

For co-immunoprecipitation:

• Polyclonal antibodies may be advantageous for pulling down protein complexes

• Monoclonal antibodies may provide more consistent results for quantitative co-IP studies

For specialized applications:

• Recombinant monoclonal antibodies (83439-1-PBS) offer advantages for applications requiring conjugation or matched antibody pairs

• Polyclonal antibodies may be better suited for detecting post-translationally modified forms of POLR2H

When selecting between polyclonal and monoclonal POLR2H antibodies, researchers should consider their experimental goals, required specificity, and the particular application to achieve optimal results .

What antigen retrieval methods are optimal for POLR2H immunohistochemistry experiments?

Effective antigen retrieval is crucial for successful POLR2H detection in fixed tissue samples. Based on validated protocols:

Primary recommended method:

• TE buffer (10 mM Tris, 1 mM EDTA) at pH 9.0

• Heat-induced epitope retrieval using pressure cooker or microwave

• Maintain retrieval solution at 95-100°C for 15-20 minutes

• Allow sections to cool slowly to room temperature (approximately 20 minutes)

Alternative method:

• Citrate buffer (10 mM sodium citrate) at pH 6.0

• Heat-induced epitope retrieval as described above

• May be preferred for certain tissue types or fixation conditions

Optimization considerations:

• Fixation time affects retrieval efficiency; tissues fixed longer may require extended retrieval

• Fresh frozen sections typically require milder retrieval conditions than FFPE tissues

• Test multiple retrieval conditions on serial sections to determine optimal protocol

Tissue-specific notes:

• Human breast cancer tissue has been specifically validated for POLR2H IHC

• Different tissue types may require adjusted retrieval conditions

• Consider tissue-specific positive controls to validate staining patterns

Protocol development:

• Start with the recommended TE buffer pH 9.0 method

• If results are suboptimal, try the alternative citrate buffer method

• Adjust retrieval time in 5-minute increments to optimize signal-to-noise ratio

• Document conditions systematically for reproducibility

The choice of antigen retrieval method significantly impacts the quality of POLR2H detection in immunohistochemistry, making systematic optimization essential for reliable results .

What controls should I include when using POLR2H antibodies in experimental workflows?

Incorporating appropriate controls is essential for ensuring reliable and interpretable results when working with POLR2H antibodies. A comprehensive control strategy includes:

Positive controls:

• Cell line controls: HeLa cells, MCF-7 cells, and HL-60 cells have been validated to express POLR2H

• Tissue controls: Mouse kidney, mouse/rat liver tissues show reliable POLR2H expression

• Recombinant protein: Purified POLR2H protein at known concentrations for standard curves

Negative controls:

• Antibody controls: Isotype-matched IgG from the same species (rabbit IgG for most POLR2H antibodies)

• Secondary antibody only: Omit primary antibody to assess secondary antibody background

• POLR2H-depleted samples: siRNA knockdown or CRISPR knockout samples when available

Application-specific controls:

For Western blotting:

• Molecular weight marker to confirm 17 kDa band size

• Loading control (e.g., GAPDH, β-actin) for normalization

• Gradient of lysate concentrations to establish linear detection range

For immunoprecipitation:

• Input control (5-10% of pre-IP lysate)

• IgG control precipitation

• Beads-only control to identify non-specific binding to matrix

For immunohistochemistry/immunofluorescence:

• Known positive tissue section with established staining pattern

• Blocking peptide competition to demonstrate specificity

• Autofluorescence control (for fluorescence applications)

For ELISA and bead array applications:

• Standard curve using recombinant POLR2H

• Blank wells (no sample)

• Dilution series to establish assay linearity

Systematic inclusion of these controls helps distinguish specific signals from artifacts, enables accurate quantification, and provides crucial validation of experimental results across different POLR2H antibody applications .

How should I troubleshoot weak or absent signal in POLR2H Western blot experiments?

When facing weak or absent POLR2H signals in Western blotting, a systematic troubleshooting approach is necessary. Consider the following strategies:

Sample preparation issues:

• Insufficient protein extraction: POLR2H is a nuclear protein; ensure lysis buffer can access nuclear proteins

• Protein degradation: Add fresh protease inhibitors; keep samples on ice; avoid repeated freeze-thaw cycles

• Insufficient protein loading: Increase total protein amount (start with 30-50 μg per lane)

• Verify protein transfer efficiency with reversible staining (Ponceau S)

Antibody-related factors:

• Suboptimal antibody dilution: Try more concentrated antibody solutions (1:200-1:500)

• Antibody degradation: Check antibody storage conditions (-20°C, avoid repeated freeze-thaw)

• Batch variability: Test new antibody lot against previous successful lot

• Wrong secondary antibody: Ensure secondary matches host species (rabbit for most POLR2H antibodies)

Detection system considerations:

• Insufficient exposure time: Increase exposure time incrementally

• ECL reagent sensitivity: Try high-sensitivity ECL substrates for weak signals

• Membrane choice: PVDF membranes may provide better protein retention than nitrocellulose

Protocol optimization:

• Blocking conditions: Test different blocking agents (5% milk vs. 3-5% BSA)

• Incubation time: Extend primary antibody incubation to overnight at 4°C

• Washing stringency: Reduce wash stringency if signal is weak (lower salt, less detergent)

POLR2H-specific considerations:

• Expected molecular weight: Confirm you're looking at the correct 17 kDa region

• Positive control inclusion: Run HeLa or MCF-7 cell lysate as a positive control

• Sample type validation: Ensure POLR2H antibody has been validated for your sample type

Systematic documentation of each troubleshooting step will help identify the critical variables affecting POLR2H detection and establish reliable protocols for consistent results .

What emerging applications are being developed for POLR2H antibodies in research?

POLR2H antibody applications are expanding beyond traditional protein detection methods to address emerging research questions. Key developments include:

In multiplex systems:

• Development of matched antibody pairs for cytometric bead arrays allows quantitative detection of POLR2H in complex biological samples

• Conjugation-ready formats (PBS only, BSA and azide free) enable custom labeling for multiplex imaging applications

• Integration into multiplex protein detection platforms for systems biology approaches

In transcription complex analysis:

• Use in ChIP-seq experiments to map POLR2H binding sites genome-wide

• Application in proximity ligation assays to study RNA polymerase complex assembly dynamics

• Combined with mass spectrometry for comprehensive identification of POLR2H interaction partners

In disease research:

• Potential biomarker applications based on altered POLR2H expression or modification in disease states

• Use in tissue microarrays to evaluate POLR2H expression across large sample cohorts

• Application in single-cell analysis platforms to examine cell-to-cell variability in transcription machinery

In mechanistic studies:

• Combined with CRISPR-based gene editing to study the functional impact of POLR2H mutations

• Application in live-cell imaging when used with appropriate fluorescent tags

• Integration with structural biology approaches to understand the role of POLR2H in polymerase complex architecture

These emerging applications represent exciting opportunities for researchers to leverage POLR2H antibodies in addressing fundamental questions about transcriptional regulation and its dysregulation in disease contexts .

How can I integrate POLR2H antibody-based experiments with other molecular biology techniques?

Integrating POLR2H antibody-based detection with complementary molecular techniques creates powerful experimental workflows for comprehensive analysis:

Multi-level expression analysis:

• Combine Western blot protein detection with RT-qPCR for POLR2H mRNA quantification

• Correlate protein levels detected by POLR2H antibodies with transcriptome data from RNA-seq

• Use immunofluorescence with RNA-FISH to simultaneously detect protein localization and associated transcripts

Functional analysis integration:

• Follow CRISPR-mediated POLR2H modification with antibody-based phenotypic assessment

• Combine chromatin immunoprecipitation (ChIP) using POLR2H antibodies with DNA sequencing

• Use POLR2H immunoprecipitation followed by mass spectrometry to identify interacting partners

Spatial and temporal dynamics:

• Integrate immunohistochemistry with spatial transcriptomics for tissue-level analysis

• Combine time-course Western blot analysis with live-cell imaging of transcription dynamics

• Use proximity ligation assays with POLR2H antibodies to study protein-protein interactions in situ

Multi-omics approaches:

• Correlate POLR2H antibody-based proteomics with epigenomic data (e.g., ATAC-seq, histone ChIP-seq)

• Integrate POLR2H binding site identification with metabolomic changes during transcriptional regulation

• Combine POLR2H complex immunoprecipitation with RNA-seq to identify associated RNAs

Protocol integration considerations:

• Sample preparation must be compatible across techniques (fixation, lysis conditions)

• Antibody specificity becomes even more critical in integrated workflows

• Consider sequential analyses from the same samples to reduce variability