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ATP2A2 / Sarcoplasmic/endoplasmic reticulum calcium ATPase 2 · Western blot design guide
Design a Western Blot for ATP2A2
Real validated ATP2A2 Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-ATP2A2 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
ATP2A2 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 | ~114.8 kDa | |
| Gel | 8–10% (standard starting point) | |
| Positive control | Cerebellum (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 | |
| Caveat | Phosphorylation-state controls | |
| Gene-set association | MSigDB Hallmark membership | |
| Isoform | 5 isoform(s) |
Section 1
Real Curated ATP2A2 Western Blot Protocols
The M01176 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 M01176) |
| Gel % | 8–10% (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 | M01176; 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 ATP2A2 Western Blot Band Size?
ATP2A2 is predicted at 114.8 kDa; isoforms could affect migration, but distinct bands and an empirical apparent mass are not demonstrated.
| Band near 114.8 kDa | Consistent with the predicted full-length ATP2A2 mass; identity needs confirmation |
| Several discrete bands | Could reflect annotated isoforms 1–5, although distinct migration is unverified |
| Weak band in whole-cell lysate | Recovery of this multi-pass membrane protein may be limited |
| Stronger band in an ER or SR membrane fraction | Consistent with ATP2A2 membrane localization; identity needs confirmation |
💡Expected ATP2A2 appearanceFull-length ATP2A2 is predicted at 114.8 kDa, but no empirical band size or isoform-specific migration is supplied; confirm any candidate band with ordinary identity controls.
| Predicted full-length mass | Provides a 114.8 kDa reference, not a measured band position |
| Splice isoform 1 | Apparent size relative to the other isoforms is unknown |
| Splice isoform 2 | Could differ in size; its mass is not supplied |
| Splice isoform 3 | Could differ in size; its mass is not supplied |
| Splice isoform 4 | Could differ in size; its mass is not supplied |
| Splice isoform 5 | Could differ in size; its mass is not supplied |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | Incomplete recovery of membrane-localized ATP2A2 is possible | Check membrane extraction and test an ER or SR membrane fraction |
| Band higher than expected | Apparent migration or band identity is uncertain | Check the size marker and confirm identity with an independent antibody or ATP2A2-depleted control |
| Band lower than expected | An isoform is possible, but no isoform size is supplied | Confirm identity and assess which ATP2A2 isoforms the sample expresses |
| Multiple bands | Five isoforms are annotated, but their separation on a blot is unverified | Compare isoform expression and validate bands with an independent antibody or ATP2A2-depleted control |
| Weak or no signal | Membrane extraction may yield little ATP2A2 | Check extraction and loading, then compare with a membrane-enriched sample |
Experiment evidence
Western blot images showing ATP2A2 band
M01176 · ATP2A2
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for ATP2A2 Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for ATP2A2 in Western blot, you can use cerebellum lysate, which shows high HPA 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: ATP2A2 is a membrane protein, so membrane-enriched lysate may improve detection.
HPA tissue expression evidence for ATP2A2
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 | Purkinje cells - cytoplasm/membrane | High | Protein (IHC) | HPA → |
| Heart muscle | cardiomyocytes | High | Protein (IHC) | HPA → |
| Skeletal muscle | myocytes | High | Protein (IHC) | HPA → |
| Bronchus | respiratory epithelial cells | Medium | Protein (IHC) | HPA → |
| Caudate | neuronal cells | 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 → |
| Bone marrow | hematopoietic cells | Not detected | Protein (IHC) | HPA → |
| Breast | adipocytes | Not detected | Protein (IHC) | HPA → |
| Cervix | glandular cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced ATP2A2 Western Blot Tips
Deeper troubleshooting and optimisation questions for ATP2A2, answered from its protein features.
What band size should I expect for ATP2A2?
Band shift · The canonical sequence has a predicted mass of 114.8 kDa. No observed Western blot band size is supplied. Use 114.8 kDa as a reference, but do not assign an unexpected apparent mass to a specific modification without further evidence.
Could ATP2A2 isoforms produce different bands?
Isoforms · Five isoforms are listed. Isoform 4 lacks residues 155–181; isoforms 2, 3, and 5 replace canonical residues 994–1042 with different short sequences. These changes could affect band position, but the supplied features do not establish which isoforms are present or whether they resolve as separate bands.
Check its epitope against the isoform sequences. An epitope within residues 155–181 is absent from isoform 4; the canonical 994–1042 region differs in isoforms 2, 3, and 5. Antibody recognition may therefore vary by isoform.
Decide whether the measurement should include all detected isoforms or a specific band, and use the same band definition across samples. Five isoforms are listed, with sequence differences that may affect antibody recognition. The supplied features do not establish that every band represents a distinct isoform.
Check its epitope against the isoform sequences. An epitope within residues 155–181 is absent from isoform 4; the canonical 994–1042 region differs in isoforms 2, 3, and 5. Antibody recognition may therefore vary by isoform.
Decide whether the measurement should include all detected isoforms or a specific band, and use the same band definition across samples. Five isoforms are listed, with sequence differences that may affect antibody recognition. The supplied features do not establish that every band represents a distinct isoform.
Which ATP2A2 modifications matter when interpreting bands?
PTM · UniProt lists phosphoserine at 38, 531, 580, and 663; phosphothreonine at 441; and 3′-nitrotyrosine at 294 and 295. These are UniProt coordinates, which may differ from antibody or paper numbering. Their presence does not establish a visible shift or explain a particular band.
Does this guide establish induction of ATP2A2?
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 ATP2A2 Western blot?
Transfer · ATP2A2 is a 114.8 kDa multi-pass ER and sarcoplasmic reticulum membrane protein. Check transfer recovery when setting conditions for this size and membrane-associated protein; the supplied features do not identify a preferred transfer method.
How should blocking be optimized?
Blocking · Standard workflow guidance: follow the M01176 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 ATP2A2 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.
How should I interpret unexpected ATP2A2 bands?
Interpretation · Compare bands with the 114.8 kDa canonical prediction and the listed isoform changes. Check whether the antibody epitope is retained in each isoform. Phosphorylation and nitration sites are documented, but the features alone cannot assign an unexpected band to a modification or isoform.
Boster reagents
ATP2A2 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-SERCA2 ATP2A2 Rabbit Monoclonal Antibody
Cat # M01176
The catalog reports M01176, an anti-ATP2A2 (SERCA2) rabbit monoclonal antibody with reported human, mouse, and rat reactivity. Its Western blot image shows SERCA2 detection in HeLa cell lysate; the supplied evidence does not show mouse or rat samples.
Which to pick: M01176 is the only listed option. It has a Western blot image using HeLa cell lysate; check its reported reactivity against your sample species.
Source: BosterBio ATP2A2 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry P16615.
- Human Protein Atlas. ATP2A2 tissue expression.
- PMC4345262 — target-verified WB comparison
- PMC11743445 — target-verified WB comparison
- PMC5513796 — target-verified WB comparison