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RFC4 / Replication factor C subunit 4 · Western blot design guide
Design a Western Blot for RFC4
Source-linked RFC4 Western blot protocol options, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-RFC4 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
RFC4 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 | ~39.7 kDa | |
| Observed band | ~37 kDa | |
| Gel | 5–20% (catalog A07702-1) | |
| 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 | Acetylated | |
| Caveat | Observed below expected | |
| Gene-set association | MSigDB Hallmark membership | |
| Isoform | 2 isoform(s) |
Section 1
Source-Linked RFC4 Western Blot Protocol Options
The A07702-1 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | human MCF-7, human Raji, human Hela, human HepG2 (catalog A07702-1) |
| Gel % | 5–20% (catalog A07702-1) |
| Load | 30 ug; reducing conditions (catalog A07702-1) |
| Transfer | a nitrocellulose membrane at 150 mA for 50-90 minutes (catalog A07702-1) |
| Membrane | nitrocellulose membrane (catalog A07702-1) |
| Blocking | 5% non-fat milk/TBS for 1.5 hour at RT (catalog A07702-1) |
| Primary antibody | A07702-1 · 0.5 μg/mL (catalog A07702-1) |
| Primary incubation | overnight at 4°C (catalog A07702-1) |
| Secondary antibody | goat anti-rabbit IgG-HRP, 1:5000 (catalog A07702-1) |
| Secondary incubation | 1.5 hour at RT (catalog A07702-1) |
| Wash | TBS-0.1%Tween 3 times with 5 minutes each (catalog A07702-1) |
| Detection | ECL (catalog A07702-1) |
Section 2
What Is the Expected RFC4 Western Blot Band Size?
RFC4 is predicted at 39.7 kDa and observed at ~37 kDa; the cause of the difference is not established.
| Band at ~37 kDa | Empirical RFC4 band in reducing whole-cell lysates; confirm identity with antibody controls |
| Band near 39.7 kDa | Near the predicted RFC4 sequence mass; confirm identity |
| Two bands near the expected region | Could reflect isoforms 1 and 2, but distinct migration is unestablished |
| Single sharp band near the expected region | Compatible with RFC4; isoforms 1 and 2 need not resolve separately |
💡Expected RFC4 appearanceRFC4 has a predicted mass of 39.7 kDa and an empirical band at ~37 kDa in reducing whole-cell lysates; the cause of the difference is unestablished, so confirm identity with antibody controls.
| Predicted RFC4 mass | 39.7 kDa by sequence; the empirical band is ~37 kDa, with no established cause for the difference |
| Splice isoform 1 | Its individual mass and migration are not supplied |
| Splice isoform 2 | Its individual mass and migration are not supplied |
| Alternative splicing of isoforms 1 and 2 | May affect apparent size, but a size difference or distinct bands are not established |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | Nuclear RFC4 may be underrepresented in the sample | Check a nuclear fraction and verify sample loading |
| Band higher than expected | Identity of the higher band is unestablished | Compare with the ~37 kDa reference band and validate with an independent antibody |
| Band lower than expected | RFC4 migrates empirically at ~37 kDa versus its predicted 39.7 kDa; the cause is unestablished | Compare with the ~37 kDa reference band and verify antibody specificity |
| Multiple bands | Isoforms 1 and 2 exist, but distinct band sizes are unestablished | Use an independent antibody or isoform-specific control to identify the bands |
| Weak or no signal | Insufficient nuclear RFC4 in the loaded sample | Check loading and test a nuclear-enriched sample |
| Fragments below expected size | Sample degradation or antibody cross-reactivity; RFC4 cleavage is not documented here | Prepare fresh lysate with protease inhibitors and verify bands with an independent antibody |
Experiment evidence
Western blot images showing RFC4 band
A07702-1 · RFC4
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for RFC4 Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for RFC4 in Western blot, you can use appendix tissue, which has high HPA expression.
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 the samples.
⚠️Feasibility: No listed tissue is undetected, so use siRNA knockdown or a KO line for a clean negative control.
HPA tissue expression evidence for RFC4
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 | lymphoid tissue | High | Protein (IHC) | HPA → |
| Bone marrow | hematopoietic cells | High | Protein (IHC) | HPA → |
| Duodenum | glandular cells | High | Protein (IHC) | HPA → |
| Lymph node | germinal center cells | High | Protein (IHC) | HPA → |
| Placenta | cytotrophoblasts | High | Protein (IHC) | HPA → |
Lower expression tissues · IHC evidence, not confirmed WB-negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Epididymis | glandular cells | Low | Protein (IHC) | HPA → |
| Heart muscle | cardiomyocytes | Low | Protein (IHC) | HPA → |
| Lung | alveolar cells | Low | Protein (IHC) | HPA → |
| Seminal vesicle | glandular cells | Low | Protein (IHC) | HPA → |
| Skeletal muscle | myocytes | Low | Protein (IHC) | HPA → |
Section 3
Advanced RFC4 Western Blot Tips
Deeper troubleshooting and optimisation questions for RFC4, answered from its protein features.
How should RFC4 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 RFC4 isoforms produce different bands?
Isoforms · RFC4 has two listed isoforms. In isoform 2, residues 269–303 are replaced and residues 304–363 are missing, so its sequence differs substantially from isoform 1. The features do not establish where either isoform migrates on a blot.
Check the antibody epitope against both sequences. Residues 1–268 are shared; isoform 2 replaces residues 269–303 and lacks residues 304–363. These are UniProt coordinates, which may differ from antibody numbering.
Check the antibody epitope against both sequences. Residues 1–268 are shared; isoform 2 replaces residues 269–303 and lacks residues 304–363. These are UniProt coordinates, which may differ from antibody numbering.
Which RFC4 modifications matter when interpreting a blot?
PTM · UniProt lists N-acetylmethionine at residue 1 and N6-acetyllysine at residues 6 and 13. These are UniProt coordinates. Their presence alone does not establish a visible band shift or explain the difference between the observed and predicted masses.
Does this guide establish induction of RFC4?
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.
How should transfer be checked for RFC4?
Transfer · Standard workflow guidance: verify transfer efficiency for the intended target size before interpreting a weak signal. Use total-protein assessment and optimize transfer for the membrane, gel and apparatus; the labelled catalog values take precedence.
How should blocking be optimized?
Blocking · Standard workflow guidance: follow the A07702-1 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 I quantify RFC4 if multiple bands appear?
Quantitation · Define the band being measured and use the same band definition across samples. RFC4 has two sequence isoforms, and the reported apparent band is about 37 kDa. Do not combine additional bands with it unless their RFC4 identity has been established.
Why does RFC4 appear near 37 kDa rather than 39.7 kDa?
Interpretation · The reported apparent band is about 37 kDa, while the predicted mass is 39.7 kDa. The supplied features do not establish the cause of this difference. Confirm band identity with an RFC4-specific antibody and an appropriate control.
Check whether the antibody recognizes both isoforms: isoform 2 replaces residues 269–303 and lacks residues 304–363. Compare unexpected bands with the reported ~37 kDa band and validate their identity before assigning them to an isoform or acetylation.
Check whether the antibody recognizes both isoforms: isoform 2 replaces residues 269–303 and lacks residues 304–363. Compare unexpected bands with the reported ~37 kDa band and validate their identity before assigning them to an isoform or acetylation.
Boster reagents
RFC4 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-RFC4 Antibody Picoband®
Cat # A07702-1
A07702-1 is a human-reactive anti-RFC4 antibody with a Western blot image showing a band near 37 kDa in MCF-7, Raji, HeLa, and HepG2 whole-cell lysates; the expected size is 40 kDa. No publication evidence is supplied.
Which to pick: A07702-1 is the only listed option. Its WB image uses four human cell lysates, so it is the documented choice for those sample contexts. Performance in other species or sample types is not established here.
Source: BosterBio RFC4 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry P35249.
- Human Protein Atlas. RFC4 tissue expression.
- PMC13469972 — target-verified WB comparison
- PMC12041875 — target-verified WB comparison
- PMC13172434 — target-verified WB comparison