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- Table of Contents
RHOA / Transforming protein RhoA · Western blot design guide
Design a Western Blot for RHOA
Real validated RHOA Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-RHOA 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
RHOA 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 | ~21.8 kDa | |
| Observed band | ~22 kDa | |
| Gel | 5–20% (catalog M00207) | |
| Positive control | Adipose tissue (IHC candidate; verify WB) +4 more | |
| Negative control | Esophagus (IHC candidate; verify WB) |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Toxin-dependent O-glycosylation + Phosphorylated | |
| Caveat | Toxin-dependent glycosylation | |
| Gene-set association | MSigDB Hallmark membership | |
| Isoform | 1 isoform(s) |
Section 1
Real Curated RHOA Western Blot Protocols
The M00207 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | human Hela, human U251, human SIHA (catalog M00207) |
| Gel % | 5–20% (catalog M00207) |
| Load | 30 ug; reducing conditions (catalog M00207) |
| Transfer | a nitrocellulose membrane at 150 mA for 50-90 minutes (catalog M00207) |
| Membrane | nitrocellulose membrane (catalog M00207) |
| Blocking | 5% non-fat milk/TBS for 1.5 hour at RT (catalog M00207) |
| Primary antibody | M00207 · 1:500 (catalog M00207) |
| Primary incubation | overnight at 4°C (catalog M00207) |
| Secondary antibody | goat anti-rabbit IgG-HRP, 1:5000 (catalog M00207) |
| Secondary incubation | 1.5 hour at RT (catalog M00207) |
| Wash | TBS-0.1%Tween 3 times with 5 minutes each (catalog M00207) |
| Detection | ECL (catalog M00207) |
Section 2
What Is the Expected RHOA Western Blot Band Size?
RHOA is predicted at 21.8 kDa and observed at ~22 kDa; the small difference has no established cause in the supplied evidence.
| Band at ~22 kDa | RHOA signal consistent with the 21.8 kDa predicted protein; confirm identity with appropriate controls |
| Band slightly below the precursor | Possible cleavage of the annotated 191–193 propeptide; a visible difference is not established |
| Additional band near ~22 kDa after microbial infection | Could reflect toxin-dependent O-linked GlcNAc at Tyr34 or Thr37, or O-linked glucose at Thr37; band identity needs confirmation |
| No clear band near ~22 kDa | RHOA signal may be weak or absent in the tested sample |
💡Expected RHOA appearanceRHOA has a predicted mass of 21.8 kDa and an empirical band at ~22 kDa; the annotated propeptide and infection-dependent modifications do not establish a distinct migration pattern, so confirm band identity with controls.
| UniProt predicted mass | 21.8 kDa predicts a band close to the empirical ~22 kDa signal |
| Propeptide at residues 191–193 | Cleavage could make mature RHOA slightly smaller than its precursor; a resolved shift is unproven |
| Infection-dependent O-linked GlcNAc at Tyr34 | Photorhabdus PAU_02230 modification adds one sugar; a visible shift is unproven |
| Infection-dependent O-alpha-linked GlcNAc at Thr37 | C. novyi toxin TcdA modification adds one sugar; a visible shift is unproven |
| Infection-dependent O-linked glucose at Thr37 | C. difficile toxins TcdA and TcdB modify this site alternatively; a visible shift is unproven |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | RHOA recovery or abundance in the tested lysate may be low | Check lysate loading and extraction, then compare with a known positive lysate |
| Band higher than expected | The supplied features do not establish a larger RHOA species | Compare with a positive control and confirm identity using an independent RHOA antibody or RHOA depletion |
| Band lower than expected | The 191–193 propeptide may be cleaved, but its effect on migration is unproven | Confirm identity with an independent antibody or RHOA depletion |
| Broad smear instead of sharp band | The listed infection-dependent single-sugar sites do not establish a smear | Check transfer and sample quality, then verify RHOA specificity with an independent antibody or depletion |
| Multiple bands | No distinct isoform masses or resolved modification bands are supplied | Use RHOA depletion and a second antibody to identify the specific band |
| Weak or no signal | RHOA may be poorly recovered from the cell membrane or cytoskeleton | Check extraction and loading, and include a known positive lysate |
Experiment evidence
Western blot images showing RHOA band
M00207 · RHOA
M00207-1 · RHOA
M00207-1 · RHOA
M00207-2 · RHOA
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for RHOA Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for RHOA in Western blot, you can use adipose tissue lysate, which shows medium expression in HPA.
Positive control: Adipose tissue (IHC candidate; verify WB)
Negative control: Esophagus (IHC candidate; verify WB)
Loading controls: Run GAPDH, β-actin, and a total-protein stain such as Ponceau alongside the samples.
⚠️Feasibility: HPA reports RHOA as not detected in esophagus, making it a candidate negative tissue control.
HPA tissue expression evidence for RHOA
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 | Medium | Protein (IHC) | HPA → |
| Adrenal gland | glandular cells | Medium | Protein (IHC) | HPA → |
| Breast | adipocytes | Medium | Protein (IHC) | HPA → |
| Caudate | glial cells | Medium | Protein (IHC) | HPA → |
| Cerebellum | cells in molecular layer | Medium | Protein (IHC) | HPA → |
Lower expression tissues · IHC evidence, not confirmed WB-negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Esophagus | squamous epithelial cells | Not detected | Protein (IHC) | HPA → |
| Liver | cholangiocytes | Not detected | Protein (IHC) | HPA → |
| Oral mucosa | squamous epithelial cells | Not detected | Protein (IHC) | HPA → |
| Vagina | squamous epithelial cells | Not detected | Protein (IHC) | HPA → |
| Appendix | glandular cells | Low | Protein (IHC) | HPA → |
Section 3
Advanced RHOA Western Blot Tips
Deeper troubleshooting and optimisation questions for RHOA, answered from its protein features.
How should RHOA 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.
Should multiple bands be assigned to RHOA isoforms?
Isoforms · Only one isoform is listed. Do not assign extra bands to alternative isoforms based on this record alone.
Could modifications change RHOA band mobility?
PTM · The record lists modified residues, glycosylation sites, prenylation, methylation, phosphorylation, ADP-ribosylation, and ubiquitin-like conjugation. These features make modified forms plausible, but the record does not establish a specific shift size. Compare any shifted bands with the main band near 22 kDa.
Does this guide establish induction of RHOA?
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.
What transfer method to use for RHOA Western blot?
Transfer · RHOA is small, with a predicted mass of 21.8 kDa. Choose transfer conditions that retain proteins near 22 kDa, and check transfer efficiency in that region.
How should blocking be optimized?
Blocking · Standard workflow guidance: follow the M00207 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 RHOA bands be quantified?
Quantitation · Quantify the band near the observed 22 kDa position consistently across samples. Because the record lists modifications, report any additional bands separately unless their identity is established.
Why is the RHOA band near 22 kDa?
Interpretation · RHOA has a predicted mass of 21.8 kDa, consistent with the observed band near 22 kDa. Use that region as the starting point for band identification.
RHOA is listed at the cytoplasmic side of the cell membrane with a lipid anchor and also in the cytoplasm and cytoskeleton. Check both membrane-associated and cytoplasmic material when interpreting a weak or missing band.
RHOA has listed modification and ubiquitin-like conjugation features, and it interacts with other proteins. An extra band alone does not establish its identity. Keep it separate from the main approximately 22 kDa band until verified.
RHOA is listed at the cytoplasmic side of the cell membrane with a lipid anchor and also in the cytoplasm and cytoskeleton. Check both membrane-associated and cytoplasmic material when interpreting a weak or missing band.
RHOA has listed modification and ubiquitin-like conjugation features, and it interacts with other proteins. An extra band alone does not establish its identity. Keep it separate from the main approximately 22 kDa band until verified.
Boster reagents
RHOA 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 A Rabbit Monoclonal Antibody
Cat # M00207
Real WB data
Anti-Rho A + B + C Rabbit Monoclonal Antibody
Cat # M00207-1
Real WB data
Anti-Rho RHOA Rabbit Monoclonal Antibody
Cat # M00207-2
Three catalogued the supplier antibodies have Western blot images: M00207 for Rho A, M00207-1 for Rho A+B+C, and M00207-2 for Rho expression in HeLa lysate. The supplied evidence does not establish RHOA specificity for the latter two.
Which to pick: For a RHOA-focused blot, start with M00207, whose image is described as Rho A analysis. Choose M00207-1 if detecting Rho A, B, and C together is acceptable. M00207-2 has a HeLa lysate blot, but its cue does not specify RHOA.
Source: BosterBio RHOA gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry P61586.
- Human Protein Atlas. RHOA tissue expression.
- PMC3950223 — target-verified WB comparison
- PMC6830327 — target-verified WB comparison
- PMC4503437 — target-verified WB comparison