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- Table of Contents
RHOD / Rho-related GTP-binding protein RhoD · Western blot design guide
Design a Western Blot for RHOD
Real validated RHOD Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-RHOD 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
RHOD 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 | ~23.5 kDa | |
| Gel | 12–15% (standard starting point) | |
| Negative control | Suggested KO / knockdown lysate |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Methylated + Cleaved | |
| Caveat | Modification-state controls | |
| Gene-set association | MSigDB Hallmark membership | |
| Isoform | 1 isoform(s) |
Section 1
Real Curated RHOD Western Blot Protocols
The A05942 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | MCF-7, Raw264.7, H9C2 (catalog A05942) |
| 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 | A05942; 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 RHOD Western Blot Band Size?
RHOD has a predicted 23.5 kDa precursor; propeptide cleavage could slightly alter migration, but no empirical band size or visible effect is established.
| Band near 23.5 kDa | Consistent with the predicted RHOD precursor size; identity requires controls |
| Band slightly below 23.5 kDa | Could reflect removal of the 208..210 propeptide |
| Close doublet near 23.5 kDa | Could represent precursor and processed RHOD if they resolve |
| Single band near 23.5 kDa without a resolved doublet | Precursor and processed RHOD may migrate too closely to distinguish |
💡Expected RHOD appearanceUniProt predicts a 23.5 kDa RHOD precursor; removal of its three-residue propeptide may cause a small difference, but no empirical band size establishes migration, so confirm identity with controls.
| UniProt predicted mass | Places the 210-residue precursor near 23.5 kDa |
| Propeptide at residues 208..210 | Adds three residues to the precursor relative to the processed form |
| Propeptide cleavage | May yield a slightly smaller mature protein |
| Precursor and mature RHOD | May migrate too closely to resolve as separate bands |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | Membrane-associated RHOD may be poorly recovered | Check membrane extraction and include a positive lysate control |
| Band higher than expected | Identity or migration of the band is unestablished | Compare with a molecular-weight marker and verify specificity by RHOD depletion |
| Band lower than expected | Propeptide removal may slightly reduce size | Compare with the predicted precursor size and verify the band by RHOD depletion |
| Multiple bands | Precursor and processed RHOD may resolve separately, or bands may be nonspecific | Use RHOD depletion to identify responsive bands |
| Weak or no signal | Recovery of membrane-associated RHOD may be low | Check extraction and loading with a positive lysate control |
Experiment evidence
Western blot images showing RHOD band
A05942 · RHOD
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for RHOD Western blot
🧪For positive controls for RHOD in Western blot, you can use no HPA-supported tissue or cell line from the supplied evidence.
Positive control: No high/medium HPA tissue identified
Negative control: Suggested KO / knockdown lysate
Loading controls: Run GAPDH, β-actin, and a total-protein stain alongside the samples.
⚠️Feasibility: RHOD is membrane-associated, and the supplied HPA data identify no validated positive or negative sample.
HPA tissue expression evidence for RHOD
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 |
|---|---|---|---|---|
| No high/medium HPA tissues identified in the supplied evidence. | ||||
Lower expression tissues · IHC evidence, not confirmed WB-negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| No lower-expression tissue rows available in the supplied evidence. | ||||
Section 3
Advanced RHOD Western Blot Tips
Deeper troubleshooting and optimisation questions for RHOD, answered from its protein features.
How should RHOD 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 RHOD isoforms explain multiple bands?
Isoforms · The supplied record lists one isoform and no alternative sequence. Do not assign additional bands to RHOD isoforms on this evidence alone.
Which RHOD modification should inform band interpretation?
PTM · UniProt lists a cysteine methyl ester at residue 207, using UniProt numbering. It also lists prenylation as a keyword and describes RHOD as lipid anchored, but supplies no prenylation site. Keep these distinctions when interpreting bands.
No glycosylation sites are listed, and the glycosylation count is zero. The supplied features therefore give no basis to attribute a higher band to glycosylation.
No glycosylation sites are listed, and the glycosylation count is zero. The supplied features therefore give no basis to attribute a higher band to glycosylation.
Does this guide establish induction of RHOD?
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 RHOD?
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 A05942 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 RHOD be quantified?
Quantitation · Standard workflow guidance: quantify only a validated target band within the linear exposure range. Use consistent sample preparation and loading, retain biological replicates, and avoid interpreting saturation or loading differences as regulation.
Should RHOD migrate at its predicted 23.5 kDa?
Interpretation · 23.5 kDa is the predicted mass; no observed band position is supplied. RHOD has a C-terminal propeptide at UniProt residues 208–210 and a cysteine methyl ester at UniProt residue 207. These features alone do not establish a visible shift or explain any difference from predicted mass.
RHOD is listed at the cytoplasmic side of the cell membrane and at early endosomes. Check which fraction your sample contains when interpreting a weak or missing band, and compare like fractions when quantifying RHOD.
Compare them with the predicted 23.5 kDa mass while noting that no empirical band position is supplied. The listed propeptide at UniProt residues 208–210 and methyl ester at residue 207 do not, by themselves, identify an unexpected band or establish a visible shift.
RHOD is listed at the cytoplasmic side of the cell membrane and at early endosomes. Check which fraction your sample contains when interpreting a weak or missing band, and compare like fractions when quantifying RHOD.
Compare them with the predicted 23.5 kDa mass while noting that no empirical band position is supplied. The listed propeptide at UniProt residues 208–210 and methyl ester at residue 207 do not, by themselves, identify an unexpected band or establish a visible shift.
Boster reagents
RHOD 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-Rho D Antibody
Cat # A05942
The catalog reports one anti-RHOD antibody, A05942, with reported Human, Mouse, and Rat reactivity. Its WB image shows MCF-7, Raw264.7, and H9C2 cell lysates at a 1:500 antibody dilution. The supplied evidence is limited to this product record and image caption.
Which to pick: A05942 is the only listed option. Choose it if its reported reactivity and the WB examples using MCF-7, Raw264.7, and H9C2 lysates fit your experiment; those examples do not establish performance in every sample.
Source: BosterBio RHOD gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry O00212.
- Human Protein Atlas. RHOD tissue expression.
- PMC5444730 — target-verified WB comparison