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ATP1A2 / Sodium/potassium-transporting ATPase subunit alpha-2 · Western blot design guide
Design a Western Blot for ATP1A2
Source-linked ATP1A2 Western blot protocol options, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-ATP1A2 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
ATP1A2 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 | ~112.3 kDa | |
| Observed band | Approximately 112 kDa | |
| Gel | 5–20% (catalog A02064-1) | |
| Positive control | Cerebellum (IHC candidate; verify WB) +4 more | |
| Negative control | Adrenal gland (IHC candidate; verify WB) |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Phosphorylated + Cleaved | |
| Caveat | Heating-condition controls | |
| Gene-set association | MSigDB C7 membership | |
| Isoform | 1 isoform(s) |
Section 1
Source-Linked ATP1A2 Western Blot Protocol Options
The A02064-1 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | rat brain ,(100°C boiled for 5 minutes), rat brain ,(37°C incubated for 10 minutes), mouse brain ,(100°C boiled for 5 minutes) (catalog A02064-1) |
| Gel % | 5–20% (catalog A02064-1) |
| Load | 30 ug; reducing conditions (catalog A02064-1) |
| Transfer | a nitrocellulose membrane at 150 mA for 50-90 minutes (catalog A02064-1) |
| Membrane | nitrocellulose membrane (catalog A02064-1) |
| Blocking | 5% non-fat milk/TBS for 1.5 hour at RT (catalog A02064-1) |
| Primary antibody | A02064-1 · 0.5 μg/mL (catalog A02064-1) |
| Primary incubation | overnight at 4°C (catalog A02064-1) |
| Secondary antibody | goat anti-rabbit IgG-HRP, 1:5000 (catalog A02064-1) |
| Secondary incubation | 1.5 hour at RT (catalog A02064-1) |
| Wash | TBS-0.1%Tween 3 times with 5 minutes each (catalog A02064-1) |
| Detection | ECL (catalog A02064-1) |
Section 2
What Is the Expected ATP1A2 Western Blot Band Size?
ATP1A2 is predicted at 112.3 kDa and observed at approximately 112 kDa; the small difference has no established cause.
| Band at approximately 112 kDa | matches the empirical ATP1A2 band and the 112.3 kDa predicted mass |
| Band slightly below 112 kDa | could reflect removal of the annotated five-residue propeptide |
| Weak band in whole-cell lysate | may reflect limited recovery of this multi-pass membrane protein |
| Band enriched in a membrane fraction | is consistent with ATP1A2 cell-membrane localization |
💡Expected ATP1A2 appearanceATP1A2 has a predicted mass of 112.3 kDa and an empirical band at approximately 112 kDa; confirm band identity with antibody specificity controls.
| Predicted full-length mass | sets the reference size at 112.3 kDa |
| Full-length precursor | retains the annotated residues 1–5 propeptide |
| Propeptide at residues 1–5 | its removal would slightly reduce mass |
| Mature protein after propeptide removal | could migrate slightly below the full-length precursor |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | ATP1A2 is a multi-pass membrane protein and may be poorly recovered | check membrane extraction and probe a membrane-enriched fraction |
| Band higher than expected | incomplete solubilization may affect migration of this membrane protein | review solubilization conditions and confirm identity with a second antibody |
| Band lower than expected | removal of the five-residue propeptide could cause a small decrease; a larger decrease is unexplained | compare precursor and mature forms and verify identity with a second antibody |
| Multiple bands | the supplied features do not establish distinct migrating forms | verify band identity with a second antibody and appropriate controls |
| Weak or no signal | limited recovery of membrane-localized ATP1A2 | check membrane extraction and sample loading |
Experiment evidence
Western blot images showing ATP1A2 band
A02064-1 · ATP1A2
M02064 · ATP1A2
A02064 · ATP1A2
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for ATP1A2 Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for ATP1A2 in Western blot, you can use cerebellum tissue lysate, where HPA reports high expression.
Positive control: Cerebellum (IHC candidate; verify WB)
Negative control: Adrenal gland (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, ATP1A2 may require membrane-enriched lysate for a clear signal.
HPA tissue expression evidence for ATP1A2
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 | molecular layer - neuropil | High | Protein (IHC) | HPA → |
| Cerebral cortex | neuropil | High | Protein (IHC) | HPA → |
| Appendix | glandular cells | Medium | Protein (IHC) | HPA → |
| Caudate | glial cells | Medium | Protein (IHC) | HPA → |
| Duodenum | glandular cells | Medium | Protein (IHC) | HPA → |
Lower expression tissues · IHC evidence, not confirmed WB-negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adrenal gland | glandular cells | Not detected | Protein (IHC) | HPA → |
| Bone marrow | hematopoietic cells | Not detected | Protein (IHC) | HPA → |
| Breast | adipocytes | Not detected | Protein (IHC) | HPA → |
| Bronchus | respiratory epithelial cells | Not detected | Protein (IHC) | HPA → |
| Cervix | glandular cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced ATP1A2 Western Blot Tips
Deeper troubleshooting and optimisation questions for ATP1A2, answered from its protein features.
How should ATP1A2 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 isoforms explain multiple ATP1A2 bands?
Isoforms · The supplied record lists one isoform and no alternative sequence. It therefore provides no isoform-based explanation for additional bands.
Which phosphorylation sites matter when interpreting ATP1A2 bands?
PTM · UniProt lists phosphoserine at 10, 439, 450, 496, 559, 587, 672, 826 and 940, plus phosphothreonine at 570. Serine 940 is annotated as phosphorylated by PKA. These are UniProt coordinates; check the numbering convention of any antibody or paper before comparing sites. Their presence alone does not establish a visible band shift.
Does this guide establish induction of ATP1A2?
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 ATP1A2 Western blot?
Transfer · ATP1A2 is a 1020-residue, multi-pass cell-membrane protein with a predicted mass of 112.3 kDa. Check transfer efficiency around 112 kDa with a total-protein stain and adjust the transfer conditions if that region remains in the gel. These features do not specify one transfer method.
How should blocking be optimized?
Blocking · Standard workflow guidance: follow the A02064-1 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 ATP1A2 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.
Should ATP1A2 migrate above its predicted 112.3 kDa mass?
Interpretation · The reported band is approximately 112 kDa, close to the predicted 112.3 kDa. The listed modifications do not establish a visible shift or explain a mass difference; compare the band with a molecular weight marker.
UniProt annotates a propeptide at residues 1–5. Account for this annotation when checking an antibody epitope near the N terminus. The supplied evidence does not show that processing produces a resolvable second band.
Compare the approximately 112 kDa band using equal sample loading and a consistent membrane-protein preparation. ATP1A2 is a multi-pass membrane protein, so differences in membrane recovery can affect its signal. A total-ATP1A2 band does not by itself measure phosphorylation at the listed sites.
The supplied record lists one isoform and an N-terminal propeptide at residues 1–5; it does not establish the identity of a distinct lower band. Check whether the antibody recognizes the relevant ATP1A2 sequence and whether the band tracks the approximately 112 kDa signal across samples.
UniProt annotates a propeptide at residues 1–5. Account for this annotation when checking an antibody epitope near the N terminus. The supplied evidence does not show that processing produces a resolvable second band.
Compare the approximately 112 kDa band using equal sample loading and a consistent membrane-protein preparation. ATP1A2 is a multi-pass membrane protein, so differences in membrane recovery can affect its signal. A total-ATP1A2 band does not by itself measure phosphorylation at the listed sites.
The supplied record lists one isoform and an N-terminal propeptide at residues 1–5; it does not establish the identity of a distinct lower band. Check whether the antibody recognizes the relevant ATP1A2 sequence and whether the band tracks the approximately 112 kDa signal across samples.
Boster reagents
ATP1A2 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-ATP1A2 Antibody Picoband®
Cat # A02064-1
Real WB data
Anti-ATP1A2 Rabbit Monoclonal Antibody
Cat # M02064
Real WB data
Anti-ATP1A2 Antibody
Cat # A02064
Three the supplier anti-ATP1A2 antibodies have Western blot images: A02064-1 in rat and mouse brain lysates, M02064 in human fetal heart lysate, and A02064 in COS7, HepG2, and Jurkat lysates with a peptide-blocked lane. These examples document tested specimens, not universal validation.
Which to pick: Choose by specimen and reported reactivity: A02064-1 for rat or mouse brain, M02064 for human fetal heart, or A02064 for COS7, HepG2, or Jurkat comparisons. All three have WB images; A02064 alone lists Monkey reactivity.
Source: BosterBio ATP1A2 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry P50993.
- Human Protein Atlas. ATP1A2 tissue expression.
- PMC8824814 — target-verified WB comparison
- PMC8607279 — target-verified WB comparison
- PMC3865589 — target-verified WB comparison