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- Table of Contents
RPA3 / Replication protein A 14 kDa subunit · Western blot design guide
Design a Western Blot for RPA3
Source-linked RPA3 Western blot protocol options, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-RPA3 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
RPA3 Western Blot Experimental Design Guide
Expected bands, source-linked protocol options, controls and antibodies — the at-a-glance facts below, then the full design guide.
Must know before running
| Expected band | ~13.6 kDa | |
| Observed band | ~17 kDa | |
| Gel | 12% (catalog A04696-2) | |
| Positive control | Adipose tissue (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 | Acetylated + Ubl conjugation | |
| Caveat | Modification-state controls | |
| Gene-set association | MSigDB Hallmark membership | |
| Isoform | 1 isoform(s) |
Section 1
Source-Linked RPA3 Western Blot Protocol Options
The A04696-2 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | human K562, human Jurkat, human Hela (catalog A04696-2) |
| Gel % | 12% (catalog A04696-2) |
| Load | 30 ug; reducing conditions (catalog A04696-2) |
| Transfer | a nitrocellulose membrane at 150 mA for 50-90 minutes (catalog A04696-2) |
| Membrane | nitrocellulose membrane (catalog A04696-2) |
| Blocking | 5% non-fat milk/TBS for 1.5 hour at RT (catalog A04696-2) |
| Primary antibody | A04696-2 · 0.5 μg/mL (catalog A04696-2) |
| Primary incubation | overnight at 4°C (catalog A04696-2) |
| Secondary antibody | goat anti-rabbit IgG-HRP, 1:5000 (catalog A04696-2) |
| Secondary incubation | 1.5 hour at RT (catalog A04696-2) |
| Wash | TBS-0.1%Tween 3 times with 5 minutes each (catalog A04696-2) |
| Detection | ECL (catalog A04696-2) |
Section 2
What Is the Expected RPA3 Western Blot Band Size?
RPA3 is predicted at 13.6 kDa and observed near 17 kDa on a reducing Western blot; the cause of the difference is not established.
| Band near 17 kDa in whole-cell lysate | Matches the empirical RPA3 band; confirm identity with controls. |
| Band near 14 kDa | Near the 13.6 kDa predicted mass; identity requires confirmation. |
| Band near 17 kDa in a nuclear fraction | Consistent with RPA3's nuclear location and empirical migration. |
| Little signal in a cytoplasmic fraction | Consistent with RPA3's nuclear location; check the nuclear fraction. |
💡Expected RPA3 appearanceRPA3 has a predicted mass of 13.6 kDa, while antibody QC shows a band at approximately 17 kDa in reducing whole-cell lysates; confirm band identity with ordinary controls.
| UniProt predicted mass | Sets a 13.6 kDa sequence-based reference; the empirical band is approximately 17 kDa. |
| 121-residue sequence | Underlies the predicted mass; no separate migration effect is established. |
| N-acetylvaline at residue 2 | Its effect on apparent band size is not established. |
| RPA heterotrimer membership | No separate band-size effect under the supplied reducing conditions is established. |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | Nuclear RPA3 may be poorly recovered. | Check nuclear extraction and a positive-control lysate. |
| Band higher than expected | RPA3 migrates near 17 kDa in the supplied QC blot, above its 13.6 kDa prediction; the cause is unknown. | Compare with the QC band and verify identity using an independent antibody or RPA3 depletion. |
| Band lower than expected | The band may be a fragment or an unrelated signal; no cleavage is listed. | Check sample integrity and verify identity by RPA3 depletion. |
| Multiple bands | Additional bands have no established isoform explanation in the supplied record. | Compare their response to RPA3 depletion and check antibody specificity. |
| Weak or no signal | Low RPA3 recovery or insufficient detection is possible. | Check loading, transfer and a positive-control lysate. |
| Fragments below expected size | Sample degradation is possible; no processed fragment is listed. | Repeat with fresh, protected lysate and verify fragment identity. |
Experiment evidence
Western blot images showing RPA3 band
A04696-2 · RPA3
A04696-1 · RPA3
A04696 · RPA3
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for RPA3 Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for RPA3 in Western blot, you can use adipose tissue lysate, which has high HPA expression.
Positive control: Adipose tissue (IHC candidate; verify WB)
Negative control: Suggested KO / knockdown lysate
Loading controls: Run GAPDH, β-actin, and a total-protein stain alongside the samples.
⚠️Feasibility: No clean negative tissue is documented; use siRNA knockdown or a KO line.
HPA tissue expression evidence for RPA3
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 |
|---|---|---|---|---|
| Adipose tissue | adipocytes | High | Protein (IHC) | HPA → |
| Adrenal gland | glandular cells | High | Protein (IHC) | HPA → |
| Appendix | glandular cells | High | Protein (IHC) | HPA → |
| Bone marrow | hematopoietic cells | High | Protein (IHC) | HPA → |
| Breast | adipocytes | High | Protein (IHC) | HPA → |
Lower expression tissues · IHC evidence, not confirmed WB-negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Esophagus | squamous epithelial cells | Medium | Protein (IHC) | HPA → |
| Liver | cholangiocytes | Medium | Protein (IHC) | HPA → |
| Lymph node | germinal center cells | Medium | Protein (IHC) | HPA → |
| Oral mucosa | squamous epithelial cells | Medium | Protein (IHC) | HPA → |
| Salivary gland | glandular cells | Medium | Protein (IHC) | HPA → |
Section 3
Advanced RPA3 Western Blot Tips
Deeper troubleshooting and optimisation questions for RPA3, answered from its protein features.
How should RPA3 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.
Could RPA3 isoforms explain multiple bands?
Isoforms · The supplied record lists one isoform and no alternative sequence. It therefore provides no isoform-based explanation for multiple RPA3 bands.
What modification is listed for RPA3?
PTM · UniProt lists N-acetylvaline at position 2. Keep that coordinate as UniProt numbering when comparing with antibody or paper descriptions. The record does not show that this modification produces a visible band shift.
Does this guide establish induction of RPA3?
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 RPA3 Western blot?
Transfer · RPA3 is a small protein with a predicted mass of 13.6 kDa. Choose and verify transfer conditions that retain proteins in this size range; check the membrane and post-transfer gel if the band is weak. The supplied features do not specify a particular membrane or transfer protocol.
How should blocking be optimized?
Blocking · Standard workflow guidance: follow the A04696-2 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 RPA3 be quantified?
Quantitation · Standard workflow guidance: quantify only a validated target band within the linear exposure range. Use consistent sample preparation and loading, retain biological replicates, and avoid interpreting saturation or loading differences as regulation.
Why might RPA3 appear near 17 kDa instead of 13.6 kDa?
Interpretation · RPA3 has a predicted mass of 13.6 kDa, while the supplied observed band is approximately 17 kDa. Its listed N-acetylvaline at UniProt position 2 does not establish the cause of that difference. Use an appropriate molecular weight marker and confirm band identity before attributing the difference to a modification.
RPA3 is listed as nuclear and as a component of RPA complexes. Compare equivalent sample fractions and loading amounts when quantifying its band. A change in signal alone does not establish a change in total RPA3 abundance if sample preparation differs.
First assess whether the approximately 17 kDa band is reproducible and specific to RPA3. The record lists one isoform, no glycosylation sites, and N-acetylvaline at UniProt position 2. These features alone do not identify additional bands or establish why the observed band differs from the predicted 13.6 kDa mass.
RPA3 is listed as nuclear and as a component of RPA complexes. Compare equivalent sample fractions and loading amounts when quantifying its band. A change in signal alone does not establish a change in total RPA3 abundance if sample preparation differs.
First assess whether the approximately 17 kDa band is reproducible and specific to RPA3. The record lists one isoform, no glycosylation sites, and N-acetylvaline at UniProt position 2. These features alone do not identify additional bands or establish why the observed band differs from the predicted 13.6 kDa mass.
Boster reagents
RPA3 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-RPA3 Antibody Picoband®
Cat # A04696-2
Real WB data
Anti-RPA3 Antibody
Cat # A04696-1
Real WB data
Anti-RPA14 RPA3 Antibody
Cat # A04696
Three the supplier anti-RPA3 antibodies have WB images. A04696-2 shows human K562, Jurkat, and HeLa lysates, with a band near 17 kDa versus 14 kDa expected. A04696-1 shows mouse spleen; A04696’s WB caption does not identify the lysate.
Which to pick: Choose A04696-2 for a documented human cell lysate example or A04696-1 for a mouse spleen example. A04696-1 lists human, mouse, and rat reactivity; A04696 lists human and mouse. Their images do not document every listed species.
Source: BosterBio RPA3 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry P35244.
- Human Protein Atlas. RPA3 tissue expression.
- PMC5661258 — target-verified WB comparison
- PMC13207498 — target-verified WB comparison
- PMC2615627 — target-verified WB comparison