POLR2D Antibody

For successful experiments with POLR2D antibodies, researchers should follow these application-specific protocols and conditions:

Western Blot (WB) Protocol:

• Use 25 μg of protein lysate per lane

• Dilute primary POLR2D antibody 1:1000-1:2000 (optimize based on antibody source)

• Block with 3% nonfat dry milk in TBST

• Use HRP-conjugated anti-rabbit secondary antibody at 1:10,000 dilution

• Expected band: 16-20 kDa

Immunoprecipitation (IP) Protocol:

• Use 0.5-4.0 μg antibody for 200-400 μg of cell extract

• Precipitate overnight at 4°C with gentle rotation

• Use protein A/G beads for pull-down

• Verify by Western blot using the same or different POLR2D antibody

Immunohistochemistry (IHC) Protocol:

• Dilute antibody 1:50-1:500

• For antigen retrieval, use TE buffer pH 9.0 or citrate buffer pH 6.0

• Validated positive control: mouse testis tissue

Immunocytochemistry (ICC) Protocol:

• Dilute antibody 1:10-1:100

• Fix cells with 4% paraformaldehyde

• Validated cell line: A549

• Secondary antibody: fluorophore-conjugated anti-rabbit IgG

For all applications, researchers should store antibodies at -20°C and avoid repeated freeze-thaw cycles. Most commercial POLR2D antibodies are supplied in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 .

When facing inconsistent results with POLR2D antibodies, researchers should systematically evaluate these common issues:

For Western Blot issues:

• Multiple bands or incorrect molecular weight: POLR2D should appear at 16-20 kDa. If observing bands at other weights, consider:

  • Increasing antibody specificity by optimizing dilution (try 1:2000-1:6000)

  • Using fresh lysates (POLR2D may be susceptible to degradation)

  • Verifying extraction protocol compatibility with nuclear proteins

• Weak or no signal:

  • Reduce antibody dilution (1:500-1:1000)

  • Increase protein loading (25-50 μg)

  • Extend primary antibody incubation time (overnight at 4°C)

  • Verify sample preparation maintains POLR2D integrity

For Immunoprecipitation issues:

• Poor pull-down efficiency:

  • Increase antibody amount (4 μg for 200-400 μg lysate)

  • Extend incubation time with antibody (overnight at 4°C)

  • Optimize wash stringency

  • Consider cross-linking antibody to beads for reduced background

For Immunohistochemistry/Immunocytochemistry issues:

• High background or non-specific staining:

  • Optimize blocking (try 5% BSA or goat serum)

  • Reduce primary antibody concentration (1:200-1:500)

  • Ensure proper antigen retrieval (compare TE buffer pH 9.0 vs. citrate buffer pH 6.0)

  • Include appropriate negative controls

• Weak or no signal:

  • Verify fixation conditions (overfixation can mask epitopes)

  • Increase antibody concentration (1:50-1:100)

  • Extend incubation time (overnight at 4°C)

For all applications, store antibodies properly at -20°C, avoid repeated freeze-thaw cycles, and verify antibody activity with known positive controls before troubleshooting experimental samples.

What is the significance of POLR2D in exon definition and pre-mRNA processing?

Research utilizing phosphorylation-specific antibodies against RNA Polymerase II complexes has revealed important insights into POLR2D's role in exon definition and pre-mRNA processing:

• Exon-specific localization: ChIP-seq analysis using the pCTD-2ndS2 antibody (which recognizes phosphoserine at position 2 of the second heptapeptide repeat) demonstrated predominant localization of RNA polymerase II to exonic regions of genes. This suggests a specific phosphorylation pattern associated with exon definition machinery .

• Functional implications: The preferential association of phosphorylated Pol II with exons indicates that specific phosphorylation patterns may serve as recognition signals for the splicing machinery. This creates a mechanistic link between transcription and RNA processing .

• Potential regulatory mechanism: The finding that phosphoserine at position 2 of the second repeat correlates with exon definition suggests a "phosphorylation code" that may regulate co-transcriptional splicing events. This represents an important layer of regulation beyond the primary sequence of DNA .

Further investigation of POLR2D within the RNA Pol II complex, particularly in relation to its association with splicing factors and its potential role in coordinating transcription with pre-mRNA processing, represents an exciting frontier in understanding gene expression regulation.

How can POLR2D antibodies be used to investigate transcriptional complexes and nuclear bodies?

POLR2D antibodies have proven valuable for investigating the formation and function of transcriptional complexes and nuclear bodies: