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- Table of Contents
RHEB / GTP-binding protein Rheb · Western blot design guide
Design a Western Blot for RHEB
Real validated RHEB Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-RHEB 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
RHEB 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 | ~20.5 kDa | |
| Gel | 12–15% (standard starting point) | |
| Positive control | Appendix (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 + Methylated | |
| Caveat | Phosphorylation-state controls | |
| Gene-set association | MSigDB C7 membership | |
| Isoform | 1 isoform(s) |
Section 1
Real Curated RHEB Western Blot Protocols
The A00827-1 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | mouse brain cell lysate (catalog A00827-1) |
| 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 | A00827-1 · (A) 1, (B) 2, and (C) 4 μg/mL (catalog A00827-1) |
| 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 RHEB Western Blot Band Size?
Rheb is predicted at 20.5 kDa; propeptide cleavage and listed modifications are possible influences, but their effects on visible migration are not established.
| Band near 20.5 kDa | Consistent with the predicted mass of full-length Rheb; confirm identity with antibody controls |
| Band slightly below 20.5 kDa | Could reflect cleavage of the annotated 182–184 propeptide, though a visible difference is not established |
| Weak band in soluble lysate | Consistent with Rheb association with cytoplasmic membrane surfaces |
| Band well above 20.5 kDa | Its identity and migration require verification; the supplied features do not establish this shift |
💡Expected RHEB appearanceRheb has a predicted full-length mass of 20.5 kDa; no empirical band size is supplied, and the annotated processing and modifications do not establish a visible shift, so confirm identity with antibody controls.
| Predicted full-length mass | Provides a 20.5 kDa reference, not a measured migration position |
| Propeptide at residues 182–184 | Cleavage would make the mature protein slightly smaller; a resolvable shift is not established |
| Phosphoserine at residue 130 | No apparent size shift is established by the supplied evidence |
| Cysteine methyl ester at residue 181 | No apparent size shift is established by the supplied evidence |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | Membrane-associated Rheb may be depleted from a soluble lysate | Check the membrane-containing fraction and a sample preparation control |
| Band higher than expected | The supplied features do not establish the cause of a large upward shift | Confirm identity with an independent Rheb antibody or RHEB depletion control |
| Band lower than expected | Cleavage of residues 182–184 may cause only a small size change; a larger difference is unexplained | Confirm identity and check sample handling for proteolysis |
| Multiple bands | One isoform is listed, and distinct migration states are not established | Compare bands using RHEB depletion or an independent antibody |
| Weak or no signal | Membrane-associated Rheb may be poorly recovered | Check extraction and loading of the membrane-containing fraction |
| Fragments below expected size | Proteolysis during sample handling is possible; the annotated propeptide does not explain large fragments | Prepare fresh lysate with protease inhibitors and confirm band identity |
Experiment evidence
Western blot images showing RHEB band
A00827-1 · RHEB
A00827 · RHEB
M00827-1 · RHEB
A00827-2 · RHEB
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for RHEB Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for RHEB in Western blot, you can use appendix tissue, which shows medium expression in HPA.
Positive control: Appendix (IHC candidate; verify WB)
Negative control: Adipose tissue (IHC candidate; verify WB)
Loading controls: Run GAPDH, β-actin, and a total-protein stain alongside the samples.
⚠️Feasibility: RHEB is lipid-anchored to membranes, so lysate preparation should recover membrane-associated protein.
HPA tissue expression evidence for RHEB
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 |
|---|---|---|---|---|
| Appendix | glandular cells | Medium | Protein (IHC) | HPA → |
| Bone marrow | hematopoietic cells | Medium | Protein (IHC) | HPA → |
| Breast | glandular cells | Medium | Protein (IHC) | HPA → |
| Cerebral cortex | neuropil | Medium | Protein (IHC) | HPA → |
| Cervix | 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 → |
| Fallopian tube | glandular cells | Not detected | Protein (IHC) | HPA → |
| Ovary | follicle cells | Not detected | Protein (IHC) | HPA → |
| Smooth muscle | smooth muscle cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced RHEB Western Blot Tips
Deeper troubleshooting and optimisation questions for RHEB, answered from its protein features.
What band size should I expect for RHEB?
Band shift · RHEB has a predicted mass of 20.5 kDa. No observed band size is supplied, so use 20.5 kDa as a reference rather than an exact migration target. The listed modifications alone do not establish a visible shift.
Could RHEB isoforms explain multiple bands?
Isoforms · Only one isoform is supplied, with no alternative sequence. These features do not support assigning multiple bands to known RHEB isoforms.
Which phosphorylation site matters when interpreting RHEB bands?
PTM · UniProt lists phosphoserine at position 130, modified by MAPKAPK5. This identifies a site to consider when interpreting a band, but does not establish that phosphorylation produces a detectable shift. Position 130 uses the supplied UniProt sequence numbering.
Does this guide establish induction of RHEB?
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 RHEB Western blot?
Transfer · RHEB is predicted to be 20.5 kDa, so choose transfer conditions and a membrane that retain small proteins, then check transfer efficiency. Its annotated membrane association and lipid anchor also make sample recovery relevant when comparing fractions.
How should blocking be optimized?
Blocking · Standard workflow guidance: follow the A00827-1 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 I quantify RHEB across samples?
Quantitation · Keep extraction and fractionation consistent: RHEB is annotated on the cytoplasmic side of endomembranes and the lysosome membrane, with a lipid anchor. Differences in recovered membrane material could change the measured signal. Compare like fractions and normalize consistently.
Could C-terminal processing affect RHEB migration?
Interpretation · UniProt lists a propeptide at positions 182–184 and a cysteine methyl ester at position 181. Consider these annotations when interpreting the C terminus, but neither establishes a resolvable band shift. Coordinates refer to the supplied 184-residue UniProt sequence and may differ from antibody or paper numbering.
Compare them with the predicted 20.5 kDa mass and check whether sample preparation recovers membrane-associated RHEB. The supplied features include one isoform, phosphoserine 130, cysteine methyl ester 181, and a propeptide at 182–184; they do not identify any unexpected band or establish a visible shift.
Compare them with the predicted 20.5 kDa mass and check whether sample preparation recovers membrane-associated RHEB. The supplied features include one isoform, phosphoserine 130, cysteine methyl ester 181, and a propeptide at 182–184; they do not identify any unexpected band or establish a visible shift.
Boster reagents
RHEB 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-GTP-binding protein Rheb Rheb Antibody
Cat # A00827-1
Real WB data
Anti-GTP-binding protein Rheb Rheb Antibody
Cat # A00827
Real WB data
Anti-RHEB Rabbit Monoclonal Antibody
Cat # M00827-1
Real WB data
Anti-GTP-binding protein Rheb RHEB Antibody
Cat # A00827-2
Four the supplier anti-RHEB antibodies have Western blot images showing mouse brain, rat heart, Raji, or A431 lysates. Each image documents a specific sample and condition; the supplied evidence does not establish performance across every listed species or tissue.
Which to pick: For mouse brain lysate, consider A00827-1; for rat heart lysate, A00827. For human Raji or A431 lysate, consider M00827-1 or A00827-2, respectively. All four have WB images; match your sample to the shown context and listed reactivity.
Source: BosterBio RHEB gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry Q15382.
- Human Protein Atlas. RHEB tissue expression.
- PMC9207673 — target-verified WB comparison