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RAD23B / Lysine-specific demethylase RAD23B · Western blot design guide
Design a Western Blot for RAD23B
Source-linked RAD23B Western blot protocol options, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-RAD23B 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
RAD23B 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 | ~43.2 kDa | |
| Gel | 12–15% (standard starting point) | |
| 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 | Phosphorylated | |
| Caveat | Peptide-block specificity control | |
| Gene-set association | MSigDB Hallmark membership | |
| Isoform | 2 isoform(s) |
Section 1
Source-Linked RAD23B Western Blot Protocol Options
The A02174-2 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | various cells (catalog A02174-2) |
| Gel % | 12–15% (standard starting point) |
| Load | 20–30 µg total protein per lane; optimize for abundance (standard starting point) |
| Transfer | Standard semi-dry transfer; verify efficiency (standard starting point) |
| Membrane | 0.45 µm PVDF (standard starting point) |
| Blocking | 5% milk or 5% BSA in TBST (standard starting point) |
| Primary antibody | A02174-2; use the WB datasheet starting dilution (standard starting point) |
| 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 RAD23B Western Blot Band Size?
RAD23B has a predicted mass of 43.2 kDa; isoforms and phosphorylation could affect migration, but no empirical band size or visible effect is established.
| Band near 43.2 kDa | Consistent with the predicted RAD23B mass; identity needs confirmation |
| Additional band at a different position | Could reflect isoforms 1 and 2 if their migration differs |
| Slightly shifted band | Could reflect phosphorylation, but a visible shift is unproven |
| Weaker nuclear fraction band in S phase | Consistent with the reported decrease in nuclear RAD23B during S phase |
💡Expected RAD23B appearanceUniProt predicts 43.2 kDa for RAD23B; no empirical band size is supplied, and any band near that position needs ordinary identity controls.
| UniProt predicted mass | Places the reference band near 43.2 kDa |
| Splice isoforms 1 and 2 | Could differ in size, but their masses and separation are unspecified |
| Phosphothreonine at residue 155 | Could affect migration; no visible shift is established |
| Phosphoserine at residue 160 | Could affect migration; no visible shift is established |
| Phosphotyrosine at residue 202 | Could affect migration; no visible shift is established |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | RAD23B signal in the tested lysate may be below detection | Check loading, antibody performance, and a positive control lysate |
| Band higher than expected | Phosphorylation could alter migration, but its effect here is unverified | Compare phosphatase-treated and untreated samples and confirm band identity |
| Band lower than expected | An alternative isoform could differ in size; its mass is unspecified | Check isoform recognition and confirm identity with an independent antibody |
| Multiple bands | Isoforms or phosphorylation could contribute, but distinct bands are unproven | Compare isoform recognition and phosphatase treatment, then verify band identity |
| Weak or no signal | Nuclear RAD23B levels decrease during S phase | Compare matched cell-cycle samples and check nuclear and cytoplasmic fractions |
Experiment evidence
Western blot images showing RAD23B band
A02174-2 · RAD23B
A02174-2 · RAD23B
A02174-2 · RAD23B
M02174 · RAD23B
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for RAD23B Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for RAD23B in Western blot, you can use adipose tissue, which shows 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: HPA lists no tissue with undetected expression, so use siRNA knockdown or a KO line as the negative control.
HPA tissue expression evidence for RAD23B
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 → |
| Breast | glandular cells | High | Protein (IHC) | HPA → |
| Bronchus | respiratory epithelial cells | High | Protein (IHC) | HPA → |
Lower expression tissues · IHC evidence, not confirmed WB-negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Bone marrow | hematopoietic cells | Low | Protein (IHC) | HPA → |
| Caudate | glial cells | Medium | Protein (IHC) | HPA → |
| Heart muscle | cardiomyocytes | Medium | Protein (IHC) | HPA → |
| Lung | alveolar cells type II | Medium | Protein (IHC) | HPA → |
| Lymph node | germinal center cells | Medium | Protein (IHC) | HPA → |
Section 3
Advanced RAD23B Western Blot Tips
Deeper troubleshooting and optimisation questions for RAD23B, answered from its protein features.
Where should the main RAD23B band appear?
Band shift · The supplied predicted mass is 43.2 kDa, so use that as a reference when inspecting the blot. No observed band position is supplied; the listed features do not establish an apparent mass or explain any difference from 43.2 kDa.
Could RAD23B isoforms produce different bands?
Isoforms · Two isoforms are listed. Isoform 2 lacks residues 1–72 relative to isoform 1, so it may run lower. Compare antibody recognition of the remaining sequence before assigning a lower band to isoform 2.
Could phosphorylation affect RAD23B band interpretation?
PTM · UniProt lists phosphothreonine at 155, 164 and 186; phosphoserine at 160, 174 and 199; and phosphotyrosine at 202. These are UniProt coordinates, which may differ from antibody or paper numbering. Their presence alone does not establish a visible shift; confirm a proposed phosphorylation-dependent band assignment experimentally.
Does this guide establish induction of RAD23B?
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 RAD23B?
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 A02174-2 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 can cell cycle affect RAD23B quantitation?
Quantitation · The supplied localization note says RAD23B is nuclear and cytoplasmic during G1, while nuclear levels decrease during S phase. Keep cell cycle conditions and sample fraction consistent when comparing band intensity, especially for nuclear extracts.
How should unexpected RAD23B bands be evaluated?
Interpretation · Check lower bands against isoform 2, which lacks residues 1–72, and assess whether the antibody recognizes that isoform. For other bands, compare controls and sample preparation before assigning an identity. The supplied features do not establish an observed band pattern.
RAD23B is listed in both nucleus and cytoplasm, with cell cycle-dependent distribution. Choose the fraction that matches the question and compare like fractions across samples; a change in nuclear signal may reflect redistribution rather than a change in total RAD23B.
RAD23B is listed as part of a complex with XPC and CETN2 and as interacting with NGLY1, PSMC1 and ATXN3. Those interactions alone do not identify a higher band. Test any proposed complex-related assignment under the blot’s actual sample preparation and detection conditions.
RAD23B is listed in both nucleus and cytoplasm, with cell cycle-dependent distribution. Choose the fraction that matches the question and compare like fractions across samples; a change in nuclear signal may reflect redistribution rather than a change in total RAD23B.
RAD23B is listed as part of a complex with XPC and CETN2 and as interacting with NGLY1, PSMC1 and ATXN3. Those interactions alone do not identify a higher band. Test any proposed complex-related assignment under the blot’s actual sample preparation and detection conditions.
Boster reagents
RAD23B 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-Rad23B Antibody
Cat # A02174-2
Real WB data
Anti-Rad23B (5H1) monoclonal Antibody
Cat # M02174
Two the supplier anti-RAD23B antibodies are listed for Western blot: polyclonal A02174-2 and monoclonal M02174. Both have WB images. A02174-2 is described only as tested in various cells; M02174’s caption names five cell lysates and a 1:1000 dilution.
Which to pick: Choose by listed reactivity: both cover human, mouse and rat; M02174 also lists monkey. For a documented WB starting point, M02174 reports K562, SW480, CHO-K1, 3T3 and COS7 lysates at 1:1000. A02174-2 also has a WB image, with less specific sample information.
Source: BosterBio RAD23B gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry P54727.
- Human Protein Atlas. RAD23B tissue expression.
- PMC12573188 — target-verified WB comparison
- PMC4417178 — target-verified WB comparison
- PMC3165083 — target-verified WB comparison