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ATP1A3 / Sodium/potassium-transporting ATPase subunit alpha-3 · Western blot design guide
Design a Western Blot for ATP1A3
Source-linked ATP1A3 Western blot protocol options, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-ATP1A3 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
ATP1A3 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 | ~111.7 kDa | |
| Observed band | ~90 kDa | |
| Gel | 5–20% (catalog A02278-1) | |
| Positive control | Cerebellum (IHC candidate; verify WB) +2 more | |
| Negative control | Adipose tissue (IHC candidate; verify WB) |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Phosphorylated | |
| Caveat | Phosphorylation-state controls | |
| Gene-set association | MSigDB Hallmark membership | |
| Isoform | 3 isoform(s) |
Section 1
Source-Linked ATP1A3 Western Blot Protocol Options
The A02278-1 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | rat brain, mouse brain (catalog A02278-1) |
| Gel % | 5–20% (catalog A02278-1) |
| Load | 30 ug; reducing conditions (catalog A02278-1) |
| Transfer | a nitrocellulose membrane at 150 mA for 50-90 minutes (catalog A02278-1) |
| Membrane | nitrocellulose membrane (catalog A02278-1) |
| Blocking | 5% non-fat milk/TBS for 1.5 hour at RT (catalog A02278-1) |
| Primary antibody | A02278-1 · 0.5 μg/mL (catalog A02278-1) |
| Primary incubation | overnight at 4°C (catalog A02278-1) |
| Secondary antibody | goat anti-rabbit IgG-HRP, 1:5000 (catalog A02278-1) |
| Secondary incubation | 1.5 hour at RT (catalog A02278-1) |
| Wash | TBS-0.1%Tween 3 times with 5 minutes each (catalog A02278-1) |
| Detection | ECL (catalog A02278-1) |
Section 2
What Is the Expected ATP1A3 Western Blot Band Size?
ATP1A3 is predicted at 111.7 kDa, but antibody QC reports approximately 90 kDa; the cause of that difference is not established.
| Band near 90 kDa | Empirical ATP1A3 band reported in rat and mouse brain lysates; confirm identity with controls |
| Band near 111.7 kDa | Near the sequence-predicted mass; identity requires confirmation |
| Several bands at different positions | Isoforms 1, 2 and 3 are possible contributors, but their migration is unknown |
| Weak or absent band in soluble fraction | ATP1A3 is a multi-pass membrane protein |
💡Expected ATP1A3 appearanceATP1A3 has a predicted mass of 111.7 kDa, while antibody QC reports a band near 90 kDa; the difference is unexplained, so confirm band identity with appropriate controls.
| UniProt predicted mass | 111.7 kDa is the sequence-based reference, not demonstrated migration |
| Isoform 1 | Apparent size relative to isoforms 2 and 3 is unknown |
| Isoform 2 | Apparent size relative to isoforms 1 and 3 is unknown |
| Isoform 3 | Apparent size relative to isoforms 1 and 2 is unknown |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | Membrane-associated ATP1A3 may be poorly recovered | Check membrane extraction and enrich the membrane fraction |
| Band higher than expected | Band identity or apparent migration is uncertain | Compare with a validated positive sample and an independent antibody |
| Band lower than expected | The reported 90 kDa band is below the 111.7 kDa prediction for an unestablished reason | Confirm identity with ATP1A3 depletion or an independent antibody |
| Multiple bands | Isoforms 1, 2 and 3 are annotated, but their band positions are unknown | Check isoform expression and confirm bands with ATP1A3 depletion |
| Weak or no signal | Recovery of the multi-pass membrane protein may be low | Optimize membrane solubilization and check a positive brain sample |
Experiment evidence
Western blot images showing ATP1A3 band
A02278-1 · ATP1A3
M02278 · ATP1A3
M02278 · ATP1A3
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for ATP1A3 Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for ATP1A3 in Western blot, you can use cerebellum tissue, where HPA reports high expression.
Positive control: Cerebellum (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: As a multi-pass membrane protein, ATP1A3 requires effective membrane-protein solubilization for detection.
HPA tissue expression evidence for ATP1A3
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 |
|---|---|---|---|---|
| Cerebellum | granular cells - cytoplasm/membrane | High | Protein (IHC) | HPA → |
| Cerebral cortex | neuropil | High | Protein (IHC) | HPA → |
| Heart muscle | cardiomyocytes | 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 → |
| Breast | adipocytes | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced ATP1A3 Western Blot Tips
Deeper troubleshooting and optimisation questions for ATP1A3, answered from its protein features.
How should ATP1A3 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 ATP1A3 isoforms produce different bands?
Isoforms · Three isoforms are listed. Relative to canonical isoform 1, isoform 2 replaces residues 1–2 (MG) with MGGWEEERNRRAT, and isoform 3 replaces them with MGSGGSDSYRIATSQ. These N-terminal differences could affect antibody recognition, but the features do not establish distinct visible bands or explain the approximately 90 kDa band.
Which ATP1A3 phosphorylation sites matter when interpreting bands?
PTM · UniProt lists phosphoserine at positions 37, 56, 218, 265, 442, and 933, plus phosphotyrosine at 548. Position 933 is annotated as phosphorylated by PKA. These are UniProt coordinates; antibody or paper numbering may differ. The listed sites alone do not establish a visible band shift.
Does this guide establish induction of ATP1A3?
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 ATP1A3 Western blot?
Transfer · ATP1A3 is a 1013-residue, multi-pass cell-membrane protein with a predicted mass of 111.7 kDa. Check transfer efficiency for the full-length protein and inspect the membrane and gel after transfer. The supplied features do not specify a particular transfer method.
How should blocking be optimized?
Blocking · Standard workflow guidance: follow the A02278-1 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 ATP1A3 be quantified across samples?
Quantitation · ATP1A3 is a cell-membrane protein, so prepare comparable membrane-containing samples and use consistent loading and transfer conditions. Quantify bands within the detector’s linear range. If multiple bands appear, define which band is being measured before comparing samples; the features alone do not identify the approximately 90 kDa band.
Why is the observed ATP1A3 band near 90 kDa instead of 111.7 kDa?
Interpretation · The supplied observed band is approximately 90 kDa, while the predicted mass is 111.7 kDa. The listed features do not establish the cause of this difference. Check the antibody’s target and compare the band across samples; do not assign it to a modification or isoform from apparent mass alone.
The record lists three isoforms and seven phosphorylated residues, but neither feature establishes the identity of an unexpected band. No signal peptide, propeptide, or glycosylation site is listed. Check whether each band tracks with ATP1A3 across samples and verify antibody specificity before assigning a cause.
The record lists three isoforms and seven phosphorylated residues, but neither feature establishes the identity of an unexpected band. No signal peptide, propeptide, or glycosylation site is listed. Check whether each band tracks with ATP1A3 across samples and verify antibody specificity before assigning a cause.
Boster reagents
ATP1A3 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-ATP1A3 Antibody Picoband®
Cat # A02278-1
Real WB data
Anti-ATP1A3 Rabbit Monoclonal Antibody
Cat # M02278
Both listed anti-ATP1A3 antibodies have WB images and report human, mouse, and rat reactivity. A02278-1 shows a ~90 kDa band in rat and mouse brain lysates, below the expected 112 kDa; M02278 shows a ~112 kDa band in four human cell lysates.
Which to pick: Choose A02278-1 when the rat or mouse brain lysate examples best match your experiment, while accounting for its observed band size. Choose M02278 for the documented human cell lysate examples. Both have WB images; the captions do not establish performance in every reported species or sample.
Source: BosterBio ATP1A3 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry P13637.
- Human Protein Atlas. ATP1A3 tissue expression.
- PMC11884411 — target-verified WB comparison
- PMC13536730 — target-verified WB comparison
- PMC9314279 — target-verified WB comparison