RHOB / Rho-related GTP-binding protein RhoB · Western blot design guide

Design a Western Blot for RHOB

Real validated RHOB Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-RHOB 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

Western blot protocol sheet for RHOB: expected band ~22.1 kDa, hero antibody A01550-3, catalog values and labelled standard workflow; separate PMC comparisons on the guide — Printable **RHOB** Western blot protocol sheet — expected band ~22.1 kDa, antibody A01550-3, controls and PMC citations. Open the full RHOB WB guide →

Navigate to: Protocols Band Controls Antibodies

RHOB 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	~22.1 kDa
	Observed band	Approximately 22 kDa
	Gel	5–20% (catalog A01550-3)
	Negative control ⓘ	Suggested KO / knockdown lysate

Important caveats

Reasons your observed band may differ from the expected size.

	PTM	Pathogen-dependent O-glycosylation + Phosphorylated
	Caveat	Modification-state controls
	Gene-set association	MSigDB Hallmark membership
	Isoform	1 isoform(s)

Section 1

Real Curated RHOB Western Blot Protocols

The A01550-3 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.

Recommended Western blot protocol parameters
Sample / lysate	human Hela, rat brain, mouse brain (catalog A01550-3)
Gel %	5–20% (catalog A01550-3)
Load	30 ug; reducing conditions (catalog A01550-3)
Transfer	a nitrocellulose membrane at 150 mA for 50-90 minutes (catalog A01550-3)
Membrane	nitrocellulose membrane (catalog A01550-3)
Blocking	5% non-fat milk/TBS for 1.5 hour at RT (catalog A01550-3)
Primary antibody	A01550-3 · 0.5 μg/mL (catalog A01550-3)
Primary incubation	overnight at 4°C (catalog A01550-3)
Secondary antibody	goat anti-rabbit IgG-HRP, 1:5000 (catalog A01550-3)
Secondary incubation	1.5 hour at RT (catalog A01550-3)
Wash	TBS-0.1%Tween 3 times with 5 minutes each (catalog A01550-3)
Detection	ECL (catalog A01550-3)

Section 2

What Is the Expected RHOB Western Blot Band Size?

RHOB is predicted at 22.1 kDa and observed at approximately 22 kDa; the supplied evidence does not establish a cause for their small difference.

What am I looking at on my blot?
Band at approximately 22 kDa	Matches the reported RHOB band and its 22.1 kDa predicted mass; confirm identity with appropriate controls.
Band slightly below the precursor position	Cleavage of the 194–196 propeptide could slightly reduce mass, but a resolvable shift is not established.
Little or no band in a soluble fraction	Lipid-anchored RHOB may remain in membrane fractions.
Additional band above approximately 22 kDa	Its identity is uncertain; the conditional sugar modifications at Tyr34 and Thr37 do not establish a visible shift.

💡Expected RHOB appearanceRHOB has a predicted mass of 22.1 kDa and an empirical band at approximately 22 kDa; confirm band identity with appropriate controls before assigning any other band to RHOB.

How each factor affects band size
Predicted RHOB mass	22.1 kDa provides the baseline; the reported band is approximately 22 kDa.
Propeptide at residues 194–196	Cleavage could slightly reduce mass, with no demonstrated resolvable shift.
O-linked GlcNAc at Tyr34 by Photorhabdus PAU_02230	This conditional modification could affect mobility; a visible shift is not established.
O-linked Glc at Thr37 by C. difficile toxins TcdA and TcdB	This infection-dependent modification could affect mobility; a visible shift is not established.

Why is my band missing or off?
Situation	Likely cause	Next action
No band in lysate	Lipid-anchored RHOB may be underrepresented after soluble extraction.	Check a membrane-enriched fraction and an RHOB-positive control.
Band higher than expected	The band is unassigned; the listed single-sugar modifications do not establish a visible shift.	Compare with an RHOB depletion control and check infection or toxin exposure.
Band lower than expected	Propeptide cleavage can remove residues 194–196, but a distinct lower band is unproven.	Confirm identity by RHOB depletion and assess whether the antibody recognizes the retained sequence.
Multiple bands	Their identities are uncertain; the listed modifications do not establish distinct bands.	Compare with RHOB depletion and an independent RHOB antibody.
Weak or no signal	Membrane-associated RHOB may be poorly recovered.	Check membrane recovery, sample loading, and a positive control.
Fragments below expected size	A distinct fragment is not established by the supplied features.	Check sample integrity and confirm any fragment with RHOB depletion.

Source: UniProt P62745 Human Protein Atlas product datasheet (A01550-3)

Experiment evidence

Western blot images showing RHOB band

A01550-3 · RHOB

A01550-1 · RHOB

← swipe / scroll for more images →

Real WB data behind the band-size expectations in this section.

Sample controls for RHOB Western blot

🧪For positive controls for RHOB in Western blot, you can use no HPA-supported sample because no positive tissue or cell data were supplied.

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 such as Ponceau alongside the samples.

⚠️Feasibility: HPA data are unavailable, so tissue controls cannot be validated; use a knockdown or KO line for a negative control.

HPA tissue expression evidence for RHOB

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 RHOB Western Blot Tips

Deeper troubleshooting and optimisation questions for RHOB, answered from its protein features.

How should RHOB 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 RHOB isoforms explain multiple bands?

Isoforms · The supplied UniProt record lists one isoform and no alternative sequence. It therefore provides no isoform-based explanation for multiple bands.

When might toxin-related RHOB modifications matter?

PTM · UniProt lists ADP-ribosylasparagine at position 41 by botulinum toxin, O-linked GlcNAc on Tyr34 by Photorhabdus PAU_02230, and O-linked Glc on Thr37 during microbial infection by C. difficile toxins TcdA and TcdB. Compare samples with the relevant exposure before assigning a band to these modifications; their presence does not establish a visible shift.

UniProt lists phosphotyrosine at position 154 and a cysteine methyl ester at position 193. It also lists a propeptide at positions 194–196. These are UniProt coordinates, which may differ from paper or antibody numbering. None alone identifies the cause of an observed band difference.

Does this guide establish induction of RHOB?

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 RHOB Western blot?

Transfer · Choose and verify transfer conditions that retain a protein near 22 kDa on the membrane. RHOB is listed at late endosome and cell membranes through a lipid anchor, so check recovery from the sampled fraction when evaluating a weak signal. The supplied features do not specify one transfer method.

How should blocking be optimized?

Blocking · Standard workflow guidance: follow the A01550-3 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 RHOB bands be quantified across samples?

Quantitation · Quantify the approximately 22 kDa band using consistent sample fractions and loading normalization. RHOB is listed at late endosome and cell membranes, in the nucleus, and at the cleavage furrow; changes in the sampled fraction can affect measured signal.

Should RHOB migrate at its predicted 22.1 kDa?

Interpretation · The reported apparent band is approximately 22 kDa, close to the predicted 22.1 kDa. RHOB has listed modifications and a propeptide at UniProt positions 194–196, but their presence alone does not establish a visible shift or explain any mass difference.

Check whether the band tracks RHOB across comparable samples and fractions before assigning an identity. The supplied record lists one isoform, several modifications, and a propeptide at UniProt positions 194–196, but these features alone do not establish that any unexpected band is RHOB or predict its apparent mass.

Boster reagents

RHOB 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

Western blot analysis of RHOB using anti-RHOB antibody (A01550-3). Electrophoresis was performed on a 5-20% SDS-PAGE gel at 70V (Stacking gel) / 90V (Resolving gel) for 2-3 hours. The sample well of each lane was loaded with 30 ug of sample under reducing conditions. Lane 1: human Hela whole cell lysates, Lane 2: rat brain tissue lysates, Lane 3: mouse brain tissue lysates. After electrophoresis, proteins were transferred to a nitrocellulose membrane at 150 mA for 50-90 minutes. Blocked the membrane with 5% non-fat milk/TBS for 1.5 hour at RT. The membrane was incubated with rabbit anti-RHOB antigen affinity purified polyclonal antibody (Catalog # A01550-3) at 0.5 μg/mL overnight at 4°C, then washed with TBS-0.1%Tween 3 times with 5 minutes each and probed with a goat anti-rabbit IgG-HRP secondary antibody at a dilution of 1:5000 for 1.5 hour at RT. The signal is developed using an Enhanced Chemiluminescent detection (ECL) kit (Catalog # EK1002) with Tanon 5200 system. A specific band was detected for RHOB at approximately 22 kDa. The expected band size for RHOB is at 22 kDa.

Anti-RHOB Antibody Picoband®

Cat # A01550-3

View product →

Real WB data

Western blot analysis of RHOB using anti-RHOB antibody (A01550-1). Electrophoresis was performed on a 5-20% SDS-PAGE gel at 70V (Stacking gel) / 90V (Resolving gel) for 2-3 hours. The sample well of each lane was loaded with 30 ug of sample under reducing conditions. Lane 1: human Hela whole cell lysates, Lane 2: rat brain tissue lysates, Lane 3: rat C6 whole cell lysates, Lane 4: mouse brain tissue lysates, Lane 5: monkey COS-7 whole cell lysates. After electrophoresis, proteins were transferred to a nitrocellulose membrane at 150 mA for 50-90 minutes. Blocked the membrane with 5% non-fat milk/TBS for 1.5 hour at RT. The membrane was incubated with rabbit anti-RHOB antigen affinity purified polyclonal antibody (Catalog # A01550-1) at 0.5 μg/mL overnight at 4°C, then washed with TBS-0.1%Tween 3 times with 5 minutes each and probed with a goat anti-rabbit IgG-HRP secondary antibody at a dilution of 1:5000 for 1.5 hour at RT. The signal is developed using an Enhanced Chemiluminescent detection (ECL) kit (Catalog # EK1002) with Tanon 5200 system. A specific band was detected for RHOB at approximately 22 kDa. The expected band size for RHOB is at 22 kDa.

Anti-RHOB Antibody Picoband®

Cat # A01550-1

View product →

Both listed anti-RHOB polyclonal antibodies have WB images showing an approximately 22 kDa band in the tested lysates. The captions report 30 µg per lane and 0.5 µg/mL primary antibody. No independent specificity validation is supplied.

Which to pick: Both have WB images with human HeLa, rat brain, and mouse brain samples. Choose A01550-1 if monkey COS-7 or rat C6 matches your sample; those examples appear only in its caption. A01550-3 covers the shared tested contexts.

Source: BosterBio RHOB gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.

References

UniProt Consortium. UniProt entry P62745.
Human Protein Atlas. RHOB tissue expression.
PMC10124901 — target-verified WB comparison
PMC4929294 — target-verified WB comparison
PMC3672819 — target-verified WB comparison