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- Table of Contents
AMOT / Angiomotin · Western blot design guide
Design a Western Blot for AMOT
Real validated AMOT Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-AMOT 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
AMOT 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 | ~118.1 kDa | |
| Observed band | ~140 kDa | |
| Gel | 5–20% (catalog A04170-1) | |
| Positive control | Colon (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 + Ubl conjugation | |
| Caveat | Phosphorylation-state controls | |
| Gene-set association | MSigDB Hallmark membership | |
| Isoform | 2 isoform(s) |
Section 1
Real Curated AMOT Western Blot Protocols
The A04170-1 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | human 293T, human Hela (catalog A04170-1) |
| Gel % | 5–20% (catalog A04170-1) |
| Load | 30 ug; reducing conditions (catalog A04170-1) |
| Transfer | a nitrocellulose membrane at 150 mA for 50-90 minutes (catalog A04170-1) |
| Membrane | nitrocellulose membrane (catalog A04170-1) |
| Blocking | 5% non-fat milk/TBS for 1.5 hour at RT (catalog A04170-1) |
| Primary antibody | A04170-1 · 0.5 μg/mL (catalog A04170-1) |
| Primary incubation | overnight at 4°C (catalog A04170-1) |
| Secondary antibody | goat anti-rabbit IgG-HRP, 1:5000 (catalog A04170-1) |
| Secondary incubation | 1.5 hour at RT (catalog A04170-1) |
| Wash | TBS-0.1%Tween 3 times with 5 minutes each (catalog A04170-1) |
| Detection | ECL (catalog A04170-1) |
Section 2
What Is the Expected AMOT Western Blot Band Size?
AMOT is predicted at 118.1 kDa, while an empirical band appears near 140 kDa; the cause of this difference is not established.
| Band near 140 kDa | Empirical AMOT band in 293T and HeLa lysates; verify identity with appropriate controls |
| Band near 118 kDa | Near the 118.1 kDa sequence-based prediction; identity requires verification |
| Several bands at different positions | Isoforms 1 and 2 are annotated, but distinct migration is unproven |
| One band without a doublet | Annotated isoforms need not appear as separately resolved bands |
💡Expected AMOT appearanceAMOT has a predicted mass of 118.1 kDa and an empirical band near 140 kDa in reducing whole-cell lysates; the difference is unexplained, so confirm band identity with appropriate controls.
| UniProt predicted mass | 118.1 kDa sequence-based reference versus the empirical band near 140 kDa; the difference is unexplained |
| Alternative splicing | Two isoforms are annotated, but separate band sizes are not established |
| Isoform 1 | Its individual mass and migration are not supplied |
| Isoform 2 | Its individual mass and migration are not supplied |
| Situation | Likely cause | Next action |
|---|---|---|
| Band higher than expected | The reported AMOT band is near 140 kDa rather than the 118.1 kDa prediction; the cause is unestablished | Compare with a validated AMOT-positive lysate and confirm identity using an independent antibody or AMOT depletion |
| Band lower than expected | An alternative isoform or protein breakdown is possible, but neither is established for that band | Confirm identity with AMOT depletion and compare antibodies recognizing different regions |
| Multiple bands | Isoforms 1 and 2 are annotated, but their band positions are unknown | Use isoform-aware antibodies or AMOT depletion to identify specific bands |
| Weak or no signal | AMOT abundance or detection may be insufficient in the tested lysate | Include a 293T or HeLa positive-control lysate and check loading and transfer |
| Fragments below expected size | Protein breakdown is possible; fragment sizes are not documented here | Use fresh lysate with protease inhibitors and confirm fragments with AMOT depletion |
Experiment evidence
Western blot images showing AMOT band
A04170-1 · AMOT
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for AMOT Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for AMOT in Western blot, you can use colon tissue, which HPA rates as highly positive.
Positive control: Colon (IHC candidate; verify WB)
Negative control: Adipose tissue (IHC candidate; verify WB)
Loading controls: Run GAPDH, β-actin, and a total-protein stain such as Ponceau alongside the samples.
⚠️Feasibility: AMOT is junction-localized, so its signal may be diluted in whole-tissue lysate.
HPA tissue expression evidence for AMOT
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 |
|---|---|---|---|---|
| Colon | endothelial cells | High | Protein (IHC) | HPA → |
| Kidney | cells in glomeruli | High | Protein (IHC) | HPA → |
| Placenta | decidual cells | High | Protein (IHC) | HPA → |
| Cerebral cortex | endothelial cells | Medium | Protein (IHC) | HPA → |
| Lung | macrophages | 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 → |
| Appendix | glandular cells | Not detected | Protein (IHC) | HPA → |
| Bone marrow | hematopoietic cells | Not detected | Protein (IHC) | HPA → |
| Bronchus | respiratory epithelial cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced AMOT Western Blot Tips
Deeper troubleshooting and optimisation questions for AMOT, answered from its protein features.
What should I check when an unexpected AMOT band appears?
Band shift · First check the antibody epitope against the two isoforms: isoform 2 lacks canonical residues 1–409. Compare the band with the expected 118.1 kDa mass and the observed approximately 140 kDa band, but do not assign its identity from size alone. UniProt also records phosphoserine 714; its presence alone does not establish a visible band shift.
How could AMOT isoforms affect band interpretation?
Isoforms · UniProt lists isoforms 1 and 2; isoform 2 lacks canonical residues 1–409. A lower band could therefore reflect isoform 2. An antibody against residues 1–409 would miss it, while one against a shared region could detect both. Confirm the antibody epitope before assigning either band.
Can phosphorylation explain an AMOT band shift?
PTM · UniProt identifies phosphoserine at position 714 in the supplied canonical sequence. That establishes a modification site, but does not show that phosphorylation produces a visible shift. If testing a phosphorylation dependent pattern, compare matched samples with and without phosphatase treatment. Keep position 714 labeled as the supplied UniProt coordinate when comparing antibody or paper numbering.
Does this guide establish induction of AMOT?
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 AMOT Western blot?
Transfer · AMOT is a relatively large protein: its predicted mass is 118.1 kDa, and an apparent band near 140 kDa has been observed. Optimize transfer conditions for this size range and check the post transfer gel and membrane to confirm recovery of the high mass band.
How should blocking be optimized?
Blocking · Standard workflow guidance: follow the A04170-1 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 AMOT bands be quantified across samples?
Quantitation · Use the same band assignment and detection conditions across samples. Because isoform 2 lacks canonical residues 1–409, establish whether the antibody recognizes both isoforms or only isoform 1 before combining band intensities. Quantify distinct bands separately unless their identities are supported.
Why might AMOT appear near 140 kDa instead of 118.1 kDa?
Interpretation · 118.1 kDa is the predicted mass, while approximately 140 kDa is an observed apparent band. AMOT has two isoforms and a documented phosphoserine, but these features alone do not establish the cause of that difference. Compare bands with isoform aware detection before assigning the 140 kDa signal.
UniProt places AMOT at tight junctions and reports cell surface localization, with possible membrane association. Check whether the chosen lysis conditions recover the AMOT containing fraction consistently across samples, especially when comparing band intensities.
UniProt places AMOT at tight junctions and reports cell surface localization, with possible membrane association. Check whether the chosen lysis conditions recover the AMOT containing fraction consistently across samples, especially when comparing band intensities.
Boster reagents
AMOT 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-Angiomotin/AMOT Antibody Picoband®
Cat # A04170-1
The catalog reports one anti-AMOT antibody, A04170-1, with a Western blot image from human 293T and HeLa whole cell lysates. The reported band is approximately 140 kDa, versus an expected 118 kDa; the supplied evidence does not resolve this difference.
Which to pick: A04170-1 is the only listed option. Its reported reactivity is human, and its Western blot image uses 293T and HeLa lysates. Consider the 140 kDa observed band versus the 118 kDa expected size when interpreting results.
Source: BosterBio AMOT gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry Q4VCS5.
- Human Protein Atlas. AMOT tissue expression.
- PMC12404752 — target-verified WB comparison
- PMC4501364 — target-verified WB comparison
- PMC4914293 — target-verified WB comparison