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- Table of Contents
HIRA / Protein HIRA · Western blot design guide
Design a Western Blot for HIRA
Real validated HIRA Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-HIRA WB antibodies. Everything you need to plan the experiment before you commit precious samples.
Evidence assembled September 2026 · For research use; verify linked source records and product datasheet before use
HIRA 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 | ~111.8 kDa | |
| Gel | 8–10% (standard starting point) | |
| Positive control | Appendix (IHC candidate; verify WB) +4 more | |
| Negative control | Suggested KO / knockdown lysate |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Phosphorylated + Ubl conjugation | |
| Caveat | Phosphorylation-state controls | |
| Gene-set association | MSigDB Hallmark membership | |
| Isoform | 2 isoform(s) |
Section 1
Real Curated HIRA Western Blot Protocols
The M03101 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | HeLa cell lysate (catalog M03101) |
| Gel % | 8–10% (standard starting point) |
| Load | 20–30 µg total protein per lane; optimize for abundance (standard starting point) |
| Transfer | Wet/tank transfer; optimize duration (standard starting point) |
| Membrane | 0.45 µm PVDF (standard starting point) |
| Blocking | 5% milk or 5% BSA in TBST (standard starting point) |
| Primary antibody | M03101; use the WB datasheet starting dilution (standard starting point) |
| Primary incubation | Overnight at 4 °C (standard starting point) |
| Secondary antibody | Species-matched HRP conjugate at validated dilution (standard starting point) |
| Secondary incubation | 1 h at room temperature (standard starting point) |
| Wash | 3 × 5 min in TBST (standard starting point) |
| Detection | ECL; bracket exposures to avoid saturation (standard starting point) |
Section 2
What Is the Expected HIRA Western Blot Band Size?
HIRA is predicted at 111.8 kDa; Long and Short isoforms could affect band patterns, but distinct migration has not been demonstrated.
| Band near 111.8 kDa | Consistent with the UniProt predicted mass; identity requires controls |
| Single band without a doublet | Long and Short isoforms may not resolve into separate bands |
| Two bands at different positions | Long and Short isoforms are possible contributors; confirm their identities |
| Upper band of a pair | Could represent the Long isoform if the isoforms resolve |
| Lower band of a pair | Could represent the Short isoform if the isoforms resolve |
💡Expected HIRA appearanceHIRA has a predicted mass of 111.8 kDa; Long and Short isoforms are annotated, but their individual masses and migration are unknown, so confirm any candidate band with identity controls.
| UniProt predicted mass | Sets a 111.8 kDa reference for the supplied sequence |
| Alternative splicing | May change apparent size; distinct bands are not established |
| Long isoform | Could migrate above Short if their sizes differ enough to resolve |
| Short isoform | Could migrate below Long if their sizes differ enough to resolve |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | HIRA is primarily nuclear and may be poorly represented in the sampled fraction | Check a nuclear fraction and a positive control |
| Band higher than expected | An isoform or an unrelated band could account for the position; no shifted mass is established | Confirm identity with HIRA knockdown and a second antibody |
| Band lower than expected | The Short isoform is possible, but its mass is not supplied | Confirm identity with isoform-specific evidence or HIRA knockdown |
| Multiple bands | Long and Short isoforms are annotated, though distinct migration is unproven | Compare bands after HIRA knockdown or with a second antibody |
| Weak or no signal | Nuclear HIRA may be underrepresented in the preparation | Enrich the nuclear fraction and verify sample loading |
Experiment evidence
Western blot images showing HIRA band
M03101 · HIRA
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for HIRA Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for HIRA in Western blot, you can use appendix tissue lysate, which HPA scores High.
Positive control: Appendix (IHC candidate; verify WB)
Negative control: Suggested KO / knockdown lysate
Loading controls: Run GAPDH, β-actin, and a total-protein stain alongside HIRA.
⚠️Feasibility: HPA lists no Not-detected tissue, so use siRNA knockdown or a KO line as the negative control.
HPA tissue expression evidence for HIRA
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.
Higher expression tissues · candidate positive controls from IHC
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Appendix | glandular cells | High | Protein (IHC) | HPA → |
| Breast | glandular cells | High | Protein (IHC) | HPA → |
| Caudate | neuronal cells | High | Protein (IHC) | HPA → |
| Cervix | glandular cells | High | Protein (IHC) | HPA → |
| Colon | endothelial cells | High | Protein (IHC) | HPA → |
Lower expression tissues · IHC evidence, not confirmed WB-negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adipose tissue | adipocytes | Medium | Protein (IHC) | HPA → |
| Adrenal gland | glandular cells | Medium | Protein (IHC) | HPA → |
| Bone marrow | hematopoietic cells | Medium | Protein (IHC) | HPA → |
| Cerebellum | cells in granular layer | Medium | Protein (IHC) | HPA → |
| Cerebral cortex | endothelial cells | Medium | Protein (IHC) | HPA → |
Section 3
Advanced HIRA Western Blot Tips
Deeper troubleshooting and optimisation questions for HIRA, answered from its protein features.
How should HIRA band migration be interpreted?
Band shift · Use the separately labelled calculated mass and catalog-observed evidence above. A sequence annotation does not establish an observed migration shift. Verify target identity with orthogonal controls.
How could HIRA isoforms affect band interpretation?
Isoforms · UniProt lists Long and Short isoforms. Short lacks residues 593–799 in canonical UniProt numbering, so it may appear lower than Long. Check whether the antibody epitope is retained before assigning either band.
Which phosphorylation sites are relevant to HIRA detection?
PTM · In canonical UniProt numbering, modified residues are Ser111, Ser549, Thr555, Ser557, Thr576, Ser584, Thr586, Ser610, Ser611, Ser612, Ser614, Ser661, Ser675 and Ser687. Thr555 is phosphorylated by CDK2. Short lacks the listed sites from Ser610 through Ser687; account for isoform coverage when choosing a phospho-specific antibody.
Does this guide establish induction of HIRA?
Induction · No general induction response is established by this guide. A pathway or gene-set association is not evidence of induction in a particular specimen. Verify the relevant treatment and control in a target-specific experiment.
What transfer method to use for HIRA Western blot?
Transfer · For canonical HIRA at 111.8 kDa, verify transfer near that size with a molecular-weight marker and membrane staining, then adjust transfer conditions if recovery is poor. The supplied features do not establish one specific method.
How should blocking be optimized?
Blocking · Standard workflow guidance: follow the M03101 datasheet where specified. Otherwise compare 5% milk or 5% BSA in TBST; for a phospho-specific assay start with BSA. Optimize background and specific signal with matched controls.
How should HIRA bands be quantified across samples?
Quantitation · Quantify Long and Short bands separately if both are resolved, and keep sample fraction and normalization consistent. HIRA is primarily nuclear and can localize to PML bodies, so changes in a nuclear fraction may reflect localization as well as abundance.
Why might HIRA migrate differently from its predicted mass?
Interpretation · Canonical HIRA is predicted at 111.8 kDa. Long and Short isoforms and documented phosphorylation complicate band assignment, but the supplied features give no observed band position. Do not attribute an apparent mass difference to either without experimental evidence.
UniProt reports HIRA localization to PML bodies immediately before senescence begins. When comparing samples near that stage, use matched fractionation and interpret changes in a nuclear or PML-enriched fraction in light of that localization.
Check whether its size is consistent with Short, which lacks canonical residues 593–799, and whether the antibody epitope remains in that isoform. No observed HIRA band position is supplied, so size alone cannot identify the band.
UniProt reports HIRA localization to PML bodies immediately before senescence begins. When comparing samples near that stage, use matched fractionation and interpret changes in a nuclear or PML-enriched fraction in light of that localization.
Check whether its size is consistent with Short, which lacks canonical residues 593–799, and whether the antibody epitope remains in that isoform. No observed HIRA band position is supplied, so size alone cannot identify the band.
Boster reagents
HIRA Western Blot Antibodies
Catalog antibodies with Western blot application and product-specific WB images. Evaluate suitability with the reported sample, controls and experimental conditions.
Real WB data
Anti-HIRA Rabbit Monoclonal Antibody
Cat # M03101
M03101 is a rabbit monoclonal anti-HIRA antibody listed for human reactivity. Its Western blot image shows HIRA expression in HeLa cell lysate; the supplied evidence does not establish performance across other samples or species.
Which to pick: Only M03101 is listed. Choose it for a human HIRA Western blot when the HeLa cell lysate example is relevant to your experiment; check suitability separately for other sample types.
Source: BosterBio HIRA gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry P54198.
- Human Protein Atlas. HIRA tissue expression.
- PMC8973876 — target-verified WB comparison
- PMC6145928 — target-verified WB comparison