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- Table of Contents
ARF5 / ADP-ribosylation factor 5 · Western blot design guide
Design a Western Blot for ARF5
Source-linked ARF5 Western blot protocol options, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-ARF5 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
ARF5 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 | ~20.5 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 | — | |
| Caveat | — | |
| Gene-set association | MSigDB C7 membership | |
| Isoform | 1 isoform(s) |
Section 1
Source-Linked ARF5 Western Blot Protocol Options
The M05021 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | Hela cell lysate (catalog M05021) |
| 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 | M05021; 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 ARF5 Western Blot Band Size?
ARF5 is predicted at 20.5 kDa; its lipid-anchor annotation does not demonstrate a migration shift, and no empirical band size is supplied.
| Single band near 20.5 kDa | Consistent with ARF5's predicted mass; confirm identity with a specific control |
| Band near 20.5 kDa in a membrane fraction | Consistent with lipid-anchored ARF5 |
| Band near 20.5 kDa in a Golgi-enriched fraction | Consistent with ARF5's Golgi localization |
| Band near 20.5 kDa in a perinuclear fraction | Consistent with ARF5's perinuclear localization |
💡Expected ARF5 appearanceARF5 has a predicted mass of 20.5 kDa, but no empirical band size is supplied; confirm a candidate band with antibody specificity and ARF5 depletion controls.
| Predicted molecular mass | Places the expected band near 20.5 kDa |
| 180-residue sequence | Underlies the predicted 20.5 kDa polypeptide mass |
| Myristate annotation | Indicates a lipid modification, with no demonstrated migration shift |
| Lipid anchor annotation | Does not establish a measurable change in apparent band size |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | Membrane-associated ARF5 may be poorly recovered | Check membrane extraction and a membrane-enriched fraction |
| Band higher than expected | Band identity or migration is uncertain | Compare with an ARF5 depletion control |
| Band lower than expected | Proteolysis or nonspecific binding is possible | Check sample handling and ARF5 depletion |
| Multiple bands | Additional bands have no established ARF5 feature-based explanation | Identify the ARF5-dependent band by depletion |
| Weak or no signal | Recovery of membrane-associated ARF5 may be low | Check extraction and antibody performance |
| Fragments below expected size | Sample degradation is possible | Use fresh lysate with protease inhibitors and confirm band identity |
Experiment evidence
Western blot images showing ARF5 band
M05021 · ARF5
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for ARF5 Western blot
🧪For positive controls for ARF5 in Western blot, you can use a sample confirmed to express ARF5; the supplied HPA evidence identifies no positive tissue or cell line.
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 provides no tissue data, so a positive sample needs independent validation and knockdown or KO is the practical negative control.
HPA tissue expression evidence for ARF5
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 ARF5 Western Blot Tips
Deeper troubleshooting and optimisation questions for ARF5, answered from its protein features.
Where should the ARF5 band appear relative to predicted mass?
Band shift · ARF5 has a predicted mass of 20.5 kDa. No empirical apparent band position is supplied, so use 20.5 kDa as a starting reference, not an exact migration requirement.
Could ARF5 isoforms explain multiple bands?
Isoforms · The supplied features list one isoform and no alternative sequence. They do not support assigning additional bands to ARF5 isoforms; check those bands independently.
Do annotated modifications prove a band shift?
PTM · The linked UniProt record describes protein features. A modification annotation alone does not demonstrate a visible shift; retain any condition or experimental qualifier attached to it.
Does this guide establish induction of ARF5?
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 ARF5?
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 M05021 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 ARF5 bands be quantified across fractions?
Quantitation · Quantify bands from comparable preparations and account for the fraction being measured. ARF5 has both membrane and perinuclear cytoplasmic localization, so a change in one fraction alone does not establish a change in total ARF5.
Does ARF5 lipid anchoring predict a visible band shift?
Interpretation · ARF5 is annotated as lipid-anchored, and Myristate appears among its keywords. These features alone do not establish a visible shift or explain a difference from the predicted 20.5 kDa.
ARF5 is reported at the Golgi apparatus, perinuclear cytoplasm, and membranes, including the trans-Golgi network membrane. When comparing samples, keep the collected fraction and preparation consistent so localization differences do not confound band intensity.
ARF5 interacts with GGA1, GGA2, and GGA3 when activated. An ARF5 band measures detected protein, not its activation state; band intensity alone cannot establish whether those interactions occurred.
Compare them with the 20.5 kDa predicted mass, but do not assign them to isoforms or lipid anchoring solely from these features. No observed band position is supplied, and the listed features do not establish why an additional band appears.
ARF5 is reported at the Golgi apparatus, perinuclear cytoplasm, and membranes, including the trans-Golgi network membrane. When comparing samples, keep the collected fraction and preparation consistent so localization differences do not confound band intensity.
ARF5 interacts with GGA1, GGA2, and GGA3 when activated. An ARF5 band measures detected protein, not its activation state; band intensity alone cannot establish whether those interactions occurred.
Compare them with the 20.5 kDa predicted mass, but do not assign them to isoforms or lipid anchoring solely from these features. No observed band position is supplied, and the listed features do not establish why an additional band appears.
Boster reagents
ARF5 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-ARF5 Rabbit Monoclonal Antibody
Cat # M05021
M05021 is an anti-ARF5 rabbit monoclonal antibody with reported human, mouse, and rat reactivity. Its Western blot image shows ARF5 expression in HeLa cell lysate. The supplied evidence does not establish Western blot performance in other specimens.
Which to pick: M05021 is the only listed option. Its HeLa cell lysate image provides a Western blot example; mouse and rat reactivity is reported, but no mouse or rat Western blot example is supplied.
Source: BosterBio ARF5 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry P84085.
- Human Protein Atlas. ARF5 tissue expression.
- PMC8399710 — target-verified WB comparison
- PMC10140647 — target-verified WB comparison
- PMC12641112 — target-verified WB comparison