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- Table of Contents
ROS1 / Proto-oncogene tyrosine-protein kinase ROS · Western blot design guide
Design a Western Blot for ROS1
Real validated ROS1 Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-ROS1 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
ROS1 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 | ~263.9 kDa | |
| Gel | 4–12% gradient (standard starting point) | |
| Negative control | Suggested KO / knockdown lysate |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Glycosylated + Phosphorylated | |
| Caveat | Modification-state controls | |
| Gene-set association | MSigDB Hallmark membership | |
| Isoform | 1 isoform(s) |
Section 1
Real Curated ROS1 Western Blot Protocols
The A04186-1 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | lysate from U-87 MG cell line, (catalog A04186-1) |
| Gel % | 4–12% gradient (standard starting point) |
| Load | 20–30 µg total protein per lane; optimize for abundance (standard starting point) |
| Transfer | Wet/tank transfer; optimize duration (standard starting point) |
| Membrane | 0.45 µm PVDF (standard starting point) |
| Blocking | 5% milk or 5% BSA in TBST (standard starting point) |
| Primary antibody | A04186-1; use the WB datasheet starting dilution (standard starting point) |
| Primary incubation | Overnight at 4 °C (standard starting point) |
| Secondary antibody | goat anti-rabbit IgG, 1:10000 (catalog A04186-1) |
| 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 ROS1 Western Blot Band Size?
ROS1’s predicted precursor mass is 263.9 kDa; cleavage and N-linked glycosylation could alter migration, but no empirical band size or migration effect is established.
| Band near 263.9 kDa | compatible with the predicted ROS1 precursor mass; confirm identity |
| Band below 263.9 kDa | could reflect cleavage of the 1–27 signal peptide |
| Band above 263.9 kDa | could reflect N-linked glycosylation; the migration effect is unmeasured |
| Broad high-mass signal | could reflect heterogeneous N-linked glycosylation; this pattern is unconfirmed |
💡Expected ROS1 appearanceROS1 has a predicted precursor mass of 263.9 kDa, but no empirical band size is supplied; signal-peptide cleavage and N-linked glycosylation may affect migration, so confirm any candidate band with identity controls.
| Predicted ROS1 precursor mass | 263.9 kDa before any migration effects |
| N-linked glycosylation at Asn52 | may increase apparent size if occupied; the shift is unmeasured |
| N-linked glycosylation at Asn114 | may increase apparent size if occupied; the shift is unmeasured |
| Signal peptide at residues 1–27 | cleavage yields a smaller mature protein than the precursor |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | membrane ROS1 may be poorly recovered | check membrane extraction and a ROS1-positive control |
| Band higher than expected | N-linked glycosylation may affect migration | compare with a validated ROS1 control and assess deglycosylation |
| Band lower than expected | signal-peptide cleavage or protein fragmentation | check band identity with another ROS1 epitope |
| Broad smear instead of sharp band | heterogeneous N-linked glycosylation is possible | compare with a deglycosylated sample and a ROS1-positive control |
| Multiple bands | processing or glycosylation states are possible | compare band recognition across ROS1 antibodies and controls |
| Weak or no signal | poor recovery of membrane ROS1 is possible | check extraction and antibody performance with a positive control |
| Fragments below expected size | protein degradation or cleavage is possible | use fresh lysate with protease inhibitors and check a second ROS1 epitope |
Experiment evidence
Western blot images showing ROS1 band
A04186-1 · ROS1
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for ROS1 Western blot
🧪For positive controls for ROS1 in Western blot, you can use a confirmed ROS1-positive sample, but the supplied HPA evidence identifies no specific 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 expression data to select tissue controls, and ROS1 is a membrane protein.
HPA tissue expression evidence for ROS1
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 ROS1 Western Blot Tips
Deeper troubleshooting and optimisation questions for ROS1, answered from its protein features.
How should ROS1 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 annotated ROS1 isoforms explain multiple bands?
Isoforms · The supplied record lists one isoform and no alternative sequence. It therefore provides no annotated isoform assignment for additional bands. Check whether each band is recognized by an antibody against a different ROS1 region before assigning its identity.
How can glycosylation affect ROS1 band interpretation?
PTM · UniProt lists 30 N-linked glycosylation sites across ROS1. If bands differ in mobility, compare untreated and deglycosylated samples to test whether glycans contribute. The site annotations do not establish which sites are occupied in your sample or predict a specific shift.
UniProt annotates phosphotyrosine at Tyr2274 and Tyr2334, both formed by autocatalysis. These are UniProt canonical-sequence coordinates; antibody or paper numbering may differ. A phospho-specific band can assess its recognized site, but these annotations alone do not establish a visible mobility shift.
Measure phospho-ROS1 and total ROS1 in matched samples so a phospho-signal change can be assessed alongside protein abundance. Tyr2274 and Tyr2334 are UniProt-annotated autophosphorylation sites; verify which site your phospho-specific antibody recognizes. Keep the quantified band consistent across samples.
UniProt annotates phosphotyrosine at Tyr2274 and Tyr2334, both formed by autocatalysis. These are UniProt canonical-sequence coordinates; antibody or paper numbering may differ. A phospho-specific band can assess its recognized site, but these annotations alone do not establish a visible mobility shift.
Measure phospho-ROS1 and total ROS1 in matched samples so a phospho-signal change can be assessed alongside protein abundance. Tyr2274 and Tyr2334 are UniProt-annotated autophosphorylation sites; verify which site your phospho-specific antibody recognizes. Keep the quantified band consistent across samples.
Does this guide establish induction of ROS1?
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 ROS1 Western blot?
Transfer · ROS1 is a 263.9 kDa single-pass membrane protein. Choose transfer conditions suitable for a protein of that size, then check both the membrane and post-transfer gel to confirm transfer. The supplied features do not specify a particular transfer method.
How should blocking be optimized?
Blocking · Standard workflow guidance: follow the A04186-1 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 ROS1 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.
Why might ROS1 migrate differently from its predicted mass?
Interpretation · ROS1 has a predicted mass of 263.9 kDa, a signal peptide at residues 1–27, and 30 annotated N-linked glycosylation sites. Processing and glycosylation may affect migration, but these features alone cannot establish a visible shift or explain a particular band. No observed band size was supplied.
The record includes a chromosomal rearrangement keyword, but supplies no fusion sequence or expected fusion mass. Its single annotated isoform also does not identify a smaller band. Use antibodies against different ROS1 regions and independent evidence before assigning the band to a rearrangement product.
The record includes a chromosomal rearrangement keyword, but supplies no fusion sequence or expected fusion mass. Its single annotated isoform also does not identify a smaller band. Use antibodies against different ROS1 regions and independent evidence before assigning the band to a rearrangement product.
Boster reagents
ROS1 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-ROS1 Antibody (C-term)
Cat # A04186-1
The catalog reports one anti-ROS1 antibody for WB: A04186-1, with reported human reactivity. Its WB image uses U-87 MG cell lysate (20 µg) at a 1:1000 primary dilution. The supplied evidence shows this sample context only.
Which to pick: A04186-1 is the only listed option and has a WB image from U-87 MG lysate. Its reported reactivity is human; assess suitability for other samples separately.
Source: BosterBio ROS1 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry P08922.
- Human Protein Atlas. ROS1 tissue expression.
- PMC11437283 — target-verified WB comparison
- PMC11310217 — target-verified WB comparison
- PMC10565643 — target-verified WB comparison