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- Table of Contents
RYBP / RING1 and YY1-binding protein · Western blot design guide
Design a Western Blot for RYBP
Source-linked RYBP Western blot protocol options, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-RYBP 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
RYBP 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 | ~24.8 kDa | |
| Gel | 12–15% (standard starting point) | |
| Positive control | Placenta (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 + Ubl conjugation | |
| Caveat | Modification-state controls | |
| Gene-set association | MSigDB C7 membership | |
| Isoform | 1 isoform(s) |
Section 1
Source-Linked RYBP Western Blot Protocol Options
The A04316 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | human A549 (lane A), HepG2 (lane B), and mouse 3T3 (lane C) cell lysates (catalog A04316) |
| Gel % | 12–15% (standard starting point) |
| Load | 20–30 µg total protein per lane; optimize for abundance (standard starting point) |
| Transfer | Short semi-dry transfer; verify retention (standard starting point) |
| Membrane | 0.45 µm PVDF (standard starting point) |
| Blocking | 5% milk or 5% BSA in TBST (standard starting point) |
| Primary antibody | A04316 · 1 μg/ml (catalog A04316) |
| 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 RYBP Western Blot Band Size?
RYBP is predicted at 24.8 kDa; phosphorylation may affect mobility, but no empirical band size or demonstrated migration shift is supplied.
| Single band near 24.8 kDa | Consistent with the predicted mass of full-length RYBP; confirm identity with controls. |
| Band with altered mobility | Phosphorylation is documented, but a visible shift is not established. |
| Stronger band in a nuclear fraction | Consistent with RYBP being primarily nuclear. |
| Weak or absent band in a cytoplasmic fraction | Consistent with primarily nuclear localization, though RYBP is also reported in the cytoplasm. |
💡Expected RYBP appearanceRYBP has a predicted mass of 24.8 kDa, but no empirical band size is supplied; use band-identity controls because its documented phosphorylation does not establish a visible shift.
| Predicted mass | Full-length RYBP is predicted at 24.8 kDa. |
| Phosphoserine at residue 99 | May affect mobility; the size of any shift is unknown. |
| Phosphoserine at residue 123 | May affect mobility; the size of any shift is unknown. |
| Phosphoserine at residue 127 | May affect mobility; the size of any shift is unknown. |
| Phosphoserine at residue 130 | May affect mobility; the size of any shift is unknown. |
| Phosphoserine at residue 227 | May affect mobility; the size of any shift is unknown. |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | RYBP is primarily nuclear and may be diluted in whole-cell lysate. | Check a nuclear fraction and include a positive-control lysate. |
| Band higher than expected | Phosphorylation may alter mobility, but the cause of a higher band is unestablished. | Compare with a phosphatase-treated sample and confirm band identity. |
| Band lower than expected | A smaller band is not explained by a listed signal peptide or propeptide. | Check sample integrity and confirm band identity. |
| Multiple bands | Different phosphorylation states are possible, but distinct bands are not established. | Compare phosphatase-treated and untreated samples and confirm band identity. |
| Weak or no signal | Primarily nuclear localization may limit signal in the sampled fraction. | Check a nuclear fraction and a positive-control lysate. |
| Fragments below expected size | Sample degradation is possible; no physiological cleavage is listed. | Prepare fresh lysate with protease inhibitors and confirm band identity. |
Experiment evidence
Western blot images showing RYBP band
A04316 · RYBP
M04316-3 · RYBP
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for RYBP Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for RYBP in Western blot, you can use placenta tissue lysate.
Positive control: Placenta (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: RYBP is primarily nuclear, so a nuclear-enriched lysate may improve detection.
HPA tissue expression evidence for RYBP
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 |
|---|---|---|---|---|
| Placenta | trophoblastic cells | High | Protein (IHC) | HPA → |
| Esophagus | squamous epithelial cells | Medium | Protein (IHC) | HPA → |
| Oral mucosa | squamous epithelial cells | Medium | Protein (IHC) | HPA → |
| Testis | sertoli cells | Medium | Protein (IHC) | HPA → |
| Tonsil | squamous epithelial cells | Medium | 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 → |
| Adrenal gland | glandular cells | Not detected | Protein (IHC) | HPA → |
| Appendix | glandular cells | Not detected | Protein (IHC) | HPA → |
| Bone marrow | hematopoietic cells | Not detected | Protein (IHC) | HPA → |
| Caudate | glial cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced RYBP Western Blot Tips
Deeper troubleshooting and optimisation questions for RYBP, answered from its protein features.
Where should the RYBP band appear?
Band shift · RYBP has a predicted mass of 24.8 kDa. No observed band position is supplied, so use 24.8 kDa as a starting point rather than an established apparent mass.
Are multiple RYBP bands expected from isoforms?
Isoforms · The supplied record lists one isoform and no alternative sequence. It therefore provides no isoform-based explanation for multiple bands.
Could phosphorylation cause a RYBP band shift?
PTM · UniProt lists phosphoserines at positions 99, 123, 127, 130, and 227. These features identify possible modification sites but do not establish a visible shift or explain any difference from the predicted 24.8 kDa mass. The positions use UniProt numbering.
Does this guide establish induction of RYBP?
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 RYBP?
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 A04316 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 RYBP be quantified across samples?
Quantitation · Keep sample preparation and the measured band consistent across samples. RYBP is primarily nuclear but is also reported in the cytoplasm, so compare like fractions when measuring changes in abundance.
What might unexpected RYBP bands mean?
Interpretation · The supplied features do not identify an observed band or establish the cause of extra bands. Check whether a band near the predicted 24.8 kDa is reproducible. The listed phosphoserines and Ubl-conjugation keyword alone cannot assign an unexpected band to a specific modification.
The supplied localization places RYBP primarily in the nucleus and also reports it in the cytoplasm. A nuclear fraction is a reasonable starting point; a cytoplasmic signal is also compatible with the record.
The supplied localization places RYBP primarily in the nucleus and also reports it in the cytoplasm. A nuclear fraction is a reasonable starting point; a cytoplasmic signal is also compatible with the record.
Boster reagents
RYBP 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-DEDAF RYBP Antibody
Cat # A04316
Real WB data
Anti-RYBP Rabbit Monoclonal Antibody
Cat # M04316-3
The catalog reports two anti-RYBP antibodies with reported Human, Mouse, and Rat reactivity. Both have Western blot images: A04316 shows human A549 and HepG2 and mouse 3T3 lysates; M04316-3 shows SW480 lysate. The captions do not describe a Rat sample.
Which to pick: For the documented human and mouse blot examples, choose A04316; its caption reports 1 μg/ml. M04316-3 has an SW480 lysate blot image. Both list Rat reactivity, but neither supplied caption shows a Rat specimen.
Source: BosterBio RYBP gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry Q8N488.
- Human Protein Atlas. RYBP tissue expression.
- PMC5331146 — target-verified WB comparison
- PMC12070685 — target-verified WB comparison
- PMC10630410 — target-verified WB comparison