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POLR2A · Western blot design guide
Design a Western Blot for POLR2A
Real validated POLR2A Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-POLR2A WB antibodies. Everything you need to plan the experiment before you commit precious samples.
Last reviewed: May 2026 · Scientific review: Boster Bio technical team
POLR2A Western Blot Experimental Design Guide
Expected bands, validated protocols, controls and antibodies — the at-a-glance facts below, then the full design guide.
Must know before running
| Expected band | ~217.2 kDa | |
| Gel | 4–12% gradient | |
| Negative control | siRNA / KO lysate |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Phosphorylated + Acetylated | |
| Caveat | Phosphorylation may shift mobility | |
| Regulation | Dna repair | |
| Isoform | 2 isoform(s) |
Section 1
Real Curated POLR2A Western Blot Protocols
Literature-validated Western blot parameters for POLR2A — gel percentage, transfer, blocking, antibody incubation and detection, extracted from published methods.
| Sample / lysate | human Hela , Lane 2: monkey COS-7 , Lane 3: human A431 , Lane 4: human PC-3 , Lane 5: human Caco-2 , Lane 6: human SW620 , Lane 7: human HEK293 , Lane 8: rat PC-12 , Lane 9: mouse testis . After Electrophoresis, proteins were transferred to a Nitrocellulose membrane at 150mA for 50-90 minutes. Blocked the membrane with 5% Non-fat Milk/ TBS for 1.5 hour at RT. The membrane was incubated with rabbit anti-POLR2A antigen affinity purified polyclonal antibody (Catalog # A01029-1) at 0.25 μg/mL overnight at 4°C, then washed with TBS-0.1%Tween 3 times with 5 minutes each and probed with a goat anti-rabbit IgG-HRP secondary antibody at a dilution of 1:5000 for 1.5 hour at RT. The signal is developed using an Enhanced Chemiluminescent detection (ECL) kit (Catalog # EK1002) with Tanon 5200 system. A specific band was detected for POLR2A at approximately 220KD. The expected band size for POLR2A is at 220KD |
| Gel % | 4–12% gradient |
| Load | 50ug |
| Transfer | nitrocellulose membrane, 150 mA, 50–90 min |
| Membrane | nitrocellulose |
| Blocking | 5% non-fat milk / TBS, 1.5 h RT |
| Primary antibody | 0.25 µg/mL |
| Primary incubation | overnight at 4 °C |
| Secondary antibody | goat anti-rabbit IgG-HRP, 1:5000 |
| Wash | TBS-0.1% Tween, 3 × 5 min |
| Detection | ECL |
| Exposure / imaging | Tanon 5200 |
| Observed band | 220 kDa |
Section 2
What Is the Expected POLR2A Western Blot Band Size?
POLR2A/RPB1 has a 217 kDa predicted backbone but often runs higher or as a doublet due to extensive CTD phosphorylation rather than glycosylation or dimerization.
| single sharp band near 217 kDa | core unmodified RPB1 backbone matching its predicted mass, since no glycosylation or cleavage is annotated |
| band running noticeably above 217 kDa, sometimes as an upper smear | heavy phosphorylation across the annotated modified residues, including CTD phosphoserines, slows migration into the hyperphosphorylated form |
| doublet with a lower and an upper band | coexistence of a hypophosphorylated pool and a hyperphosphorylated pool of RPB1 |
| signal present in both cytoplasmic and nuclear/chromatin fractions | reflects the annotated dual localization, with hypophosphorylated protein mainly cytoplasmic and hyperphosphorylated active protein nuclear and chromosome-associated |
| faint extra band at a slightly different apparent mass | expression of the second annotated splice isoform alongside the canonical isoform |
💡Expected POLR2A appearanceExpect full-length RPB1 near its 217 kDa predicted mass, often appearing as a doublet or shifted slightly higher because of extensive CTD phosphorylation, with no glycosylation, cleavage, or disulfide dimer to account for.
| Predicted mass from UniProt (217.2 kDa) | sets the baseline expected migration position for full-length RPB1 |
| Extensive phosphorylation (69 annotated modified residues, multiple phosphoserines) | shifts apparent mobility upward and underlies the classic hypophosphorylated versus hyperphosphorylated mobility difference |
| Two annotated splice isoforms | isoform 2 can contribute a minor band at a different apparent mass than isoform 1, though no isoform-specific weight is defined |
| Dual nuclear, cytoplasmic, and chromosome localization | band phosphorylation state and intensity can differ between fractions, with the hypophosphorylated pool enriched in cytoplasm and hyperphosphorylated pool nuclear/chromatin-bound |
| Absence of annotated glycosylation and disulfide bonds | rules out glyco-smearing or a roughly doubled mass band from disulfide-linked dimer formation |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | RPB1 is chromatin-associated within the nucleus, so mild lysis conditions may leave the chromatin-bound hyperphosphorylated pool insoluble | use a stringent lysis buffer with nuclease treatment or sonication to fully release nuclear and chromatin-bound RPB1 |
| Band higher than expected | extensive phosphorylation across the annotated modified residues reduces electrophoretic mobility | treat lysate with a phosphatase to collapse the shifted band toward the predicted mass and confirm a phosphorylation-driven shift |
| Multiple bands | coexistence of hypophosphorylated and hyperphosphorylated RPB1 pools plus a minor second splice isoform | run a low-percentage gel with extended separation and compare cytoplasmic versus nuclear fractions to assign each band |
| Broad smear instead of sharp band | heterogeneous phosphorylation across many annotated sites creates a continuum of mobility states | increase gel percentage or run time, or use a phospho-affinity gel to better resolve discrete phosphoforms |
| Weak or no signal | the very large size of RPB1 makes it prone to incomplete transfer | extend transfer time and use conditions optimized for high molecular weight proteins |
| Fragments below expected size | proteolytic degradation of the large 1970 amino acid protein during lysis | add protease inhibitors and keep samples cold and freshly processed |
Experiment evidence
Western blot images showing POLR2A band
A01029-1 · POLR2A
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for POLR2A Western blot
🧪For positive controls for POLR2A in Western blot, you can use HeLa cell lysate, since POLR2A is a ubiquitously expressed core RNA polymerase II subunit present in essentially all nucleated cells and tissues.
Positive control: HeLa cells
Negative control: ubiquitously expressed; use siRNA knockdown or KO line
Loading controls: Include GAPDH and β-actin antibody blots alongside a total-protein stain (stain-free gel, Ponceau S, or REVERT) to confirm equal loading.
⚠️Feasibility: No HPA expression data are available for POLR2A, and as a core, ubiquitously expressed nuclear transcription subunit it has no expected negative tissue, so specificity is best confirmed with siRNA knockdown or a CRISPR knockout cell line rather than a tissue-based negative control.
Comprehensive Human Protein Atlas IHC scoring per tissue. Rows are taken directly from the HPA tissue chart — click any row's HPA link to view the source.
Positive expression · recommended positive controls
| Tissue | Cell type | Level | Evidence | Source |
|---|
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|
Section 3
Advanced POLR2A Western Blot Tips
Deeper troubleshooting and optimisation questions for POLR2A, answered from its protein features.
Why does POLR2A run higher than its 217 kDa prediction?
RPB1 is heavily phosphorylated (69 modified residues, mostly on the CTD), and hyperphosphorylation reduces SDS-PAGE mobility. The active, nuclear form (IIO) migrates higher than the hypophosphorylated cytoplasmic form (IIA), so expect a band above 217 kDa or even two closely spaced bands depending on cell state.
Could a second POLR2A isoform appear on blots?
UniProt lists two annotated isoforms (1 and 2) arising from alternative splicing. Isoform-specific sequence differences may produce a faint secondary band near the main ~220 kDa signal. Confirm which isoform the antibody's immunogen targets before interpreting any extra band as a distinct isoform rather than a processing artifact.
Does POLR2A phosphorylation change with transcription activity?
Yes; Pol II's CTD cycles between a hypophosphorylated form, found mainly in the cytoplasm, and a hyperphosphorylated form associated with active nuclear transcription. Conditions that alter transcriptional engagement shift the IIA/IIO band ratio rather than total POLR2A abundance, so treat this as a mobility shift, not induction of new protein.
How should blocking be optimized for POLR2A detection?
Because RPB1 is a phosphoprotein, use BSA rather than milk for blocking and antibody dilution to avoid casein phosphoprotein interference, especially with phospho-CTD antibodies. Standard 5% BSA in TBST for one hour is sufficient. No glycosylation or disulfide bonds are annotated, so no reducing-agent-specific blocking changes are needed.
What transfer method to use for POLR2A Western blot?
At 217 kDa, use wet tank transfer with 0.1% SDS added to transfer buffer and extended transfer time (60-90 minutes at high voltage, or overnight at low voltage) to move this large subunit efficiently. Standard rapid semi-dry transfer often under-transfers proteins this size, giving weak or absent signal.
How should POLR2A signal be normalized for quantitation?
Normalize to total protein stain (Ponceau or stain-free) rather than a single housekeeping gene, since POLR2A phosphorylation state and nuclear/cytoplasmic distribution shift with transcriptional activity. This makes conventional loading controls like GAPDH less reliable comparators across treatments that alter transcription.
What explains extra high- or low-molecular-weight bands?
RPB1 undergoes ubiquitin-like conjugation via isopeptide bonds, which can target stalled or damaged Pol II for proteasomal degradation. This can produce higher-molecular-weight ubiquitinated smears or lower-molecular-weight degradation fragments, distinct from the IIA/IIO phosphorylation doublet, particularly after DNA damage or transcription-stress treatments.
Boster reagents
Best POLR2A Western Blot Antibodies
BosterBio's POLR2A antibodies are among the best-performing WB antibodies on the market — well cited, thoroughly validated, and orthogonally cross-validated against negative tissues and complementary methods.
Real WB data
Anti-RNA polymerase II/POLR2A Antibody Picoband®
Cat # A01029-1
Our recommended anti-POLR2A antibodies represent Boster's best-performing, highly cited reagents, thoroughly validated by Western blot and orthogonally cross-validated against negative tissue controls and complementary detection methods, giving confident, reproducible results for reliable POLR2A detection in your samples.
Which to pick: Only one Boster antibody is catalogued for POLR2A, A01029-1, which includes a real Western blot validation image (SDS-PAGE, 5-20% gel), making it the clear choice for your POLR2A Western blot experiments.
Source: BosterBio POLR2A gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry P24928.
- Human Protein Atlas. POLR2A tissue expression.