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RB1 / Retinoblastoma-associated protein · Western blot design guide
Design a Western Blot for RB1
Real validated RB1 Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-RB1 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
RB1 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 | ~106.2 kDa | |
| Observed band | ~110–120 kDa | |
| Gel | 10% (catalog A00039-3) | |
| Positive control | Adrenal gland (IHC candidate; verify WB) +4 more | |
| Negative control | Adipose tissue (IHC candidate; verify WB) |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Phosphorylated + Acetylated | |
| Caveat | Phosphorylation-state controls | |
| Gene-set association | MSigDB Hallmark membership | |
| Isoform | 1 isoform(s) |
Section 1
Real Curated RB1 Western Blot Protocols
The A00039-3 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | human HEL, human SH-SY5Y (catalog A00039-3) |
| Gel % | 10% (catalog A00039-3) |
| Load | 30 ug; reducing conditions (catalog A00039-3) |
| Transfer | a nitrocellulose membrane at 150 mA for 50-90 minutes (catalog A00039-3) |
| Membrane | nitrocellulose membrane (catalog A00039-3) |
| Blocking | 5% non-fat milk/TBS for 1.5 hour at RT (catalog A00039-3) |
| Primary antibody | A00039-3 · 1:1000 (catalog A00039-3) |
| Primary incubation | overnight at 4°C (catalog A00039-3) |
| Secondary antibody | goat anti-rabbit IgG-HRP, 1:5000 (catalog A00039-3) |
| Secondary incubation | 1.5 hour at RT (catalog A00039-3) |
| Wash | TBS-0.1%Tween 3 times with 5 minutes each (catalog A00039-3) |
| Detection | ECL (catalog A00039-3) |
Section 2
What Is the Expected RB1 Western Blot Band Size?
RB1 is predicted at 106.2 kDa and observed at ~110–120 kDa; the cause of the difference is not established.
| Band at ~110–120 kDa | Empirical RB1 band in whole-cell lysate; confirm identity with controls. |
| Band near 106.2 kDa | Near the predicted mass of full-length RB1. |
| Closely spaced bands near the RB1 region | May reflect different phosphorylation states; distinct migration is not established here. |
| Band in a nucleus-enriched fraction | Consistent with RB1 nuclear localization. |
💡Expected RB1 appearanceRB1 has a predicted mass of 106.2 kDa, while antibody QC shows a ~110–120 kDa band in whole-cell lysate; the difference is unexplained, so confirm band identity with ordinary controls.
| Predicted RB1 mass | 106.2 kDa is the sequence-based reference; the empirical band is ~110–120 kDa. |
| Phosphoserine at residue 249 | May affect apparent mobility, but no shift is established. |
| Phosphothreonine at residue 252 | May affect apparent mobility, but no shift is established. |
| Phosphoserine at residue 567 | May affect apparent mobility, but no shift is established. |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | Nuclear RB1 may be poorly recovered during extraction. | Check nuclear protein recovery and include a positive-control lysate. |
| Band higher than expected | The empirical RB1 band runs at ~110–120 kDa versus the 106.2 kDa predicted mass; the cause is unestablished. | Compare with a positive control and confirm identity by RB1 depletion. |
| Band lower than expected | No signal-peptide or propeptide cleavage is annotated to explain a smaller band. | Check molecular-weight markers and confirm identity by RB1 depletion. |
| Multiple bands | RB1 has annotated phosphorylation sites, but distinct band migration is unproven. | Compare phosphatase-treated and untreated samples, then confirm band identity. |
| Weak or no signal | RB1 recovery may vary with nuclear and cytoplasmic localization. | Check extraction and loading with compartment markers and a positive control. |
Experiment evidence
Western blot images showing RB1 band
A00039-3 · RB1
A00039-3 · RB1
M00039-1 · RB1
M00039-2 · RB1
P00039-1 · RB1
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for RB1 Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for RB1 in Western blot, you can use adrenal gland tissue, which HPA scores High.
Positive control: Adrenal gland (IHC candidate; verify WB)
Negative control: Adipose tissue (IHC candidate; verify WB)
Loading controls: Run GAPDH, β-actin, and a total-protein stain such as Ponceau alongside the samples.
⚠️Feasibility: RB1 is nuclear and sometimes cytoplasmic, so whole-cell lysates may dilute its signal.
HPA tissue expression evidence for RB1
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 → |
| Breast | glandular cells | High | Protein (IHC) | HPA → |
| Cerebellum | Purkinje cells | High | Protein (IHC) | HPA → |
| Cervix | glandular cells | High | Protein (IHC) | HPA → |
| Lymph node | non-germinal center cells | High | Protein (IHC) | HPA → |
Lower expression tissues · IHC evidence, not confirmed WB-negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adipose tissue | adipocytes | Not detected | Protein (IHC) | HPA → |
| Hippocampus | glial cells | Not detected | Protein (IHC) | HPA → |
| Caudate | neuronal cells | Low | Protein (IHC) | HPA → |
| Prostate | glandular cells | Low | Protein (IHC) | HPA → |
| Appendix | glandular cells | Medium | Protein (IHC) | HPA → |
Section 3
Advanced RB1 Western Blot Tips
Deeper troubleshooting and optimisation questions for RB1, answered from its protein features.
How should RB1 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 RB1 isoforms explain an extra band?
Isoforms · The supplied UniProt record lists one isoform and no alternative sequence. It therefore provides no sequence-based isoform explanation for an extra band. Evaluate the band without assigning it to an unlisted isoform.
Which RB1 phosphorylation sites help interpret multiple bands?
PTM · UniProt lists phosphorylation at Ser249 and Thr252 by CDK1, Ser567 by CDK2, Ser612 by CHEK1 and CHEK2, Ser807 and Ser811 by CDK1 and CDK3, Thr821 by CDK6, and Thr826 by CDK4. These are UniProt coordinates. Different phosphorylation states are plausible when assessing multiple bands, but the site list does not prove their identities.
UniProt lists N,N-dimethylproline at position 2, SMYD2-dependent methylation at Lys810 and Lys860, and PCAF-dependent acetylation at Lys873 and Lys874. Coordinates follow UniProt numbering; antibody or paper numbering may differ. These modifications are documented sites, not proof of a particular band shift.
The supplied location note places RB1 in the nucleus and describes cytoplasmic localization when hyperphosphorylated. It also says hypophosphorylated RB1 sequesters E2F1. Compare like fractions when assessing signal changes; a change in one fraction alone need not reflect a change in total RB1.
UniProt lists N,N-dimethylproline at position 2, SMYD2-dependent methylation at Lys810 and Lys860, and PCAF-dependent acetylation at Lys873 and Lys874. Coordinates follow UniProt numbering; antibody or paper numbering may differ. These modifications are documented sites, not proof of a particular band shift.
The supplied location note places RB1 in the nucleus and describes cytoplasmic localization when hyperphosphorylated. It also says hypophosphorylated RB1 sequesters E2F1. Compare like fractions when assessing signal changes; a change in one fraction alone need not reflect a change in total RB1.
Does this guide establish induction of RB1?
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 RB1?
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 A00039-3 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 RB1 signal be quantified across samples?
Quantitation · Use a consistent band region around the supplied 110–120 kDa observation and compare equivalent sample fractions. RB1 has numerous listed phosphorylation sites and a phosphorylation-related localization note, so record whether samples represent total lysate or a fraction. Do not treat a single band’s intensity as a direct measure of every RB1 form.
Why might RB1 appear above its predicted mass?
Interpretation · RB1 has a predicted mass of 106.2 kDa, while the supplied observed band is about 110–120 kDa. UniProt lists many phosphorylation and other modification sites, but their presence alone does not establish the cause of that difference or guarantee a visible shift.
Compare them with the predicted 106.2 kDa mass and the supplied 110–120 kDa observation, then consider the listed modification sites and sample fraction. The record lists one isoform and no alternative sequence. An unexpected band cannot be identified as a particular modified RB1 form from these features alone.
Compare them with the predicted 106.2 kDa mass and the supplied 110–120 kDa observation, then consider the listed modification sites and sample fraction. The record lists one isoform and no alternative sequence. An unexpected band cannot be identified as a particular modified RB1 form from these features alone.
Boster reagents
RB1 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-RB1 Antibody Picoband®
Cat # A00039-3
Real WB data
Anti-Phospho-Retinoblastoma (S780) RB1 Rabbit Monoclonal Antibody
Cat # M00039-1
Real WB data
Anti-Rb RB1 Rabbit Monoclonal Antibody
Cat # M00039-2
Real WB data
Anti-Phospho-Rb (S807) RB1 Rabbit Monoclonal Antibody
Cat # P00039-1
Real WB data
Anti-Phospho-Rb (S780) Rabbit Monoclonal Antibody
Cat # M00039S780
Five anti-RB1 antibodies have Western blot images: two for RB1 and three labeled for phosphorylation at S780 or S807. The supplied captions describe human cell lysates; one S780 image uses alkaline phosphatase treated Jurkat lysate. Mouse and rat reactivity is listed but not shown in these captions.
Which to pick: For RB1, choose A00039-3 (human, mouse, rat; HEL and SH-SY5Y images) or M00039-2 (human, mouse; Jurkat, MCF-7, 293T and K562 images). For phospho-RB1, choose M00039-1 or M00039S780 for S780, or P00039-1 for S807; check listed reactivity against your sample.
Source: BosterBio RB1 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry P06400.
- Human Protein Atlas. RB1 tissue expression.
- PMC10746934 — target-verified WB comparison
- PMC11897054 — target-verified WB comparison
- PMC7442809 — target-verified WB comparison