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- Table of Contents
RFC1 / Replication factor C subunit 1 · Western blot design guide
Design a Western Blot for RFC1
Source-linked RFC1 Western blot protocol options, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-RFC1 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
RFC1 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 | ~128.3 kDa | |
| Observed band | ~150 kDa | |
| Gel | 5–20% (catalog PA1716) | |
| Positive control | Adrenal gland (IHC candidate; verify WB) +4 more | |
| Negative control | Heart muscle (IHC candidate; verify WB) |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Phosphorylated + Ubl conjugation | |
| Caveat | Modification-state controls | |
| Gene-set association | MSigDB Hallmark membership | |
| Isoform | 2 isoform(s) |
Section 1
Source-Linked RFC1 Western Blot Protocol Options
The PA1716 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | human 293T, human Hela, human HepG2, human Raji, human K562, human CACO-2 (catalog PA1716) |
| Gel % | 5–20% (catalog PA1716) |
| Load | 30 ug; reducing conditions (catalog PA1716) |
| Transfer | a nitrocellulose membrane at 150 mA for 50-90 minutes (catalog PA1716) |
| Membrane | nitrocellulose membrane (catalog PA1716) |
| Blocking | 5% non-fat milk/TBS for 1.5 hour at RT (catalog PA1716) |
| Primary antibody | PA1716 · 0.5 μg/mL (catalog PA1716) |
| Primary incubation | overnight at 4°C (catalog PA1716) |
| Secondary antibody | goat anti-rabbit IgG-HRP, 1:5000 (catalog PA1716) |
| Secondary incubation | 1.5 hour at RT (catalog PA1716) |
| Wash | TBS-0.1%Tween 3 times with 5 minutes each (catalog PA1716) |
| Detection | ECL (catalog PA1716) |
Section 2
What Is the Expected RFC1 Western Blot Band Size?
RFC1 is predicted at 128.3 kDa and observed near 150 kDa on a reducing Western blot; the cause of the difference is not established.
| Band near 150 kDa | Empirical RFC1 band in reducing whole-cell lysates; confirm identity with controls |
| Band near 128 kDa | Near the 128.3 kDa predicted mass; identity needs confirmation |
| More than one band | Could reflect isoforms 1 and 2 if they migrate differently; distinct bands are not established |
| Weak band in a cytosolic fraction | RFC1 is localized to the nucleus |
💡Expected RFC1 appearanceRFC1 has a predicted mass of 128.3 kDa, while a reducing whole-cell blot reports a band near 150 kDa; confirm band identity with antibody and knockdown controls.
| UniProt predicted mass | Sets a 128.3 kDa sequence-based reference; the reported band is near 150 kDa |
| 1148-residue RFC1 sequence | Corresponds to the calculated 128.3 kDa polypeptide mass |
| Isoform 1 | Its individual mass and migration relative to isoform 2 are not supplied |
| Isoform 2 | Its individual mass and migration relative to isoform 1 are not supplied |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | Nuclear RFC1 may be poorly recovered during extraction | Check nuclear protein recovery and test a nuclear-enriched fraction |
| Band higher than expected | The reported RFC1 band is near 150 kDa versus a predicted 128.3 kDa; the cause is unestablished | Compare with the reported band and verify identity by RFC1 knockdown |
| Band lower than expected | An alternate isoform is possible, but its size is unknown | Check antibody epitope coverage and test band loss after RFC1 knockdown |
| Multiple bands | Isoforms 1 and 2 are annotated, but their migration is unknown | Confirm which bands respond to RFC1 knockdown |
| Weak or no signal | Poor recovery of nuclear RFC1 is possible | Assess nuclear extraction and include a positive-control lysate |
| Fragments below expected size | Sample degradation is possible; no RFC1 cleavage product is specified | Prepare fresh lysate with protease inhibitors and verify bands by RFC1 knockdown |
Experiment evidence
Western blot images showing RFC1 band
PA1716 · RFC1
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for RFC1 Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for RFC1 in Western blot, you can use adrenal gland tissue, which HPA scores High.
Positive control: Adrenal gland (IHC candidate; verify WB)
Negative control: Heart muscle (IHC candidate; verify WB)
Loading controls: Run GAPDH, β-actin, and a total-protein stain alongside the samples.
⚠️Feasibility: HPA identifies a not-detected tissue control, and RFC1's nuclear location may favor nuclear-enriched lysates.
HPA tissue expression evidence for RFC1
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 |
|---|---|---|---|---|
| Adrenal gland | glandular cells | High | Protein (IHC) | HPA → |
| Bone marrow | hematopoietic cells | High | Protein (IHC) | HPA → |
| Caudate | glial cells | High | Protein (IHC) | HPA → |
| Cerebral cortex | glial cells | High | Protein (IHC) | HPA → |
| Hippocampus | glial cells | High | Protein (IHC) | HPA → |
Lower expression tissues · IHC evidence, not confirmed WB-negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Heart muscle | cardiomyocytes | Not detected | Protein (IHC) | HPA → |
| Liver | cholangiocytes | Not detected | Protein (IHC) | HPA → |
| Skeletal muscle | myocytes | Not detected | Protein (IHC) | HPA → |
| Adipose tissue | adipocytes | Low | Protein (IHC) | HPA → |
| Epididymis | glandular cells | Low | Protein (IHC) | HPA → |
Section 3
Advanced RFC1 Western Blot Tips
Deeper troubleshooting and optimisation questions for RFC1, answered from its protein features.
How should RFC1 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 RFC1 isoforms explain distinct bands?
Isoforms · UniProt lists isoforms 1 and 2 and annotates residue 630 as missing in isoform 2. That single-residue difference would not by itself explain a large separation between bands. Check which isoform your sample and antibody can detect before assigning a second band to alternative splicing.
Which RFC1 phosphorylation sites are annotated?
PTM · UniProt coordinates list phosphotyrosine 67; phosphothreonine 110, 161 and 163; and phosphoserine 69, 71, 73, 108, 156, 164, 173, 190, 253, 281, 283, 312, 368, 537, 1104 and 1106. Use UniProt numbering when comparing sites with antibody or paper annotations. These sites do not establish a visible band shift.
Does this guide establish induction of RFC1?
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 RFC1?
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 PA1716 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 RFC1 bands be quantified?
Quantitation · RFC1 is annotated as nuclear. Compare the same identified band across samples prepared and measured consistently, and normalize within the same sample type. If multiple bands appear, quantify them separately until their identities are established; phosphorylation annotations alone cannot identify which band contains modified RFC1.
Why might RFC1 appear near 150 kDa instead of 128.3 kDa?
Interpretation · 128.3 kDa is the predicted mass, while approximately 150 kDa is the supplied apparent Western blot band. RFC1 has annotated phosphorylation sites and two isoforms, but those features alone do not establish the cause of the difference. Compare the observed band with a molecular weight marker and appropriate RFC1 controls.
First compare them with the supplied approximately 150 kDa apparent band and the 128.3 kDa predicted mass. UniProt lists two isoforms, phosphorylation, and Ubl conjugation, but supplies no evidence assigning a particular unexpected band to any of these features. Validate band identity before interpreting changes in abundance.
UniProt places RFC1 in the nucleus and identifies it as the large subunit of the RFC complex. A nuclear fraction is therefore a relevant sample to assess RFC1. Keep sample fractionation consistent across comparisons so differences in nuclear recovery are not mistaken for changes in RFC1 abundance.
First compare them with the supplied approximately 150 kDa apparent band and the 128.3 kDa predicted mass. UniProt lists two isoforms, phosphorylation, and Ubl conjugation, but supplies no evidence assigning a particular unexpected band to any of these features. Validate band identity before interpreting changes in abundance.
UniProt places RFC1 in the nucleus and identifies it as the large subunit of the RFC complex. A nuclear fraction is therefore a relevant sample to assess RFC1. Keep sample fractionation consistent across comparisons so differences in nuclear recovery are not mistaken for changes in RFC1 abundance.
Boster reagents
RFC1 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-Replication factor C subunit 1 RFC1 Antibody Picoband®
Cat # PA1716
PA1716 is a human-reactive anti-RFC1 antibody with a Western blot image from six human cell lines. The reported band is approximately 150 kDa, versus an expected 128 kDa; the supplied evidence does not establish the reason for this difference.
Which to pick: PA1716 is the only listed RFC1 antibody. Its Western blot image uses human 293T, HeLa, HepG2, Raji, K562, and CACO-2 whole-cell lysates, with antibody at 0.5 μg/mL. Use those reported conditions as a starting point for human samples.
Source: BosterBio RFC1 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry P35251.
- Human Protein Atlas. RFC1 tissue expression.
- PMC6709527 — target-verified WB comparison
- PMC11747075 — target-verified WB comparison