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- Table of Contents
ORAI3 / Protein orai-3 · Western blot design guide
Design a Western Blot for ORAI3
Source-linked ORAI3 Western blot protocol options, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-ORAI3 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
ORAI3 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 | ~31.5 kDa | |
| Gel | 12–15% (standard starting point) | |
| Positive control | Adrenal gland (IHC candidate; verify WB) +4 more | |
| Negative control | Suggested KO / knockdown lysate |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Phosphorylated | |
| Caveat | Phosphorylation-state controls | |
| Gene-set association | MSigDB C7 membership | |
| Isoform | 1 isoform(s) |
Section 1
Source-Linked ORAI3 Western Blot Protocol Options
The A09399 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | A20 cell lysate (catalog A09399) |
| Gel % | 12–15% (standard starting point) |
| Load | 20–30 µg total protein per lane; optimize for abundance (standard starting point) |
| Transfer | Standard semi-dry transfer; verify efficiency (standard starting point) |
| Membrane | 0.45 µm PVDF (standard starting point) |
| Blocking | 5% milk or 5% BSA in TBST (standard starting point) |
| Primary antibody | A09399 · (A) 1, (B) 2 and (C) 4 μg/mL (catalog A09399) |
| 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 ORAI3 Western Blot Band Size?
ORAI3 has a predicted mass of 31.5 kDa; phosphorylation and oligomerization may affect migration, but no empirical band or feature-dependent shift is established.
| Band near 31.5 kDa | Compatible with the predicted ORAI3 monomer; confirm identity with antibody controls |
| Higher molecular weight band | Could reflect retained ORAI complexes; its identity requires confirmation |
| Weak or absent band in whole-cell lysate | Membrane-associated ORAI3 may be poorly extracted |
| Possible doublet near the predicted size | Phosphorylation at Ser42 or Ser45 could affect mobility, but a visible shift is unproven |
💡Expected ORAI3 appearanceORAI3 has a predicted monomer mass of 31.5 kDa and no supplied empirical band; confirm any band near that size with antibody specificity controls, since its phosphorylation and oligomerization do not establish migration.
| Predicted ORAI3 monomer mass | Provides a 31.5 kDa reference, not a measured band position |
| Phosphoserine at Ser42 | May affect mobility; no visible shift is established |
| Phosphoserine at Ser45 | May affect mobility; no visible shift is established |
| Homomeric and heteromeric ORAI oligomerization | May produce higher molecular weight signal if complexes persist during preparation; no specific band size is established |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | Membrane-associated ORAI3 may be poorly extracted | Check membrane solubilization and test a membrane-enriched fraction |
| Band higher than expected | ORAI complexes may persist during preparation | Compare denaturing conditions and verify the band with an independent ORAI3 antibody |
| Multiple bands | Different phosphorylation states or retained complexes are possible but unconfirmed | Compare phosphatase treatment and denaturing conditions, then confirm band identity |
| Weak or no signal | Insufficient recovery of membrane-associated ORAI3 | Assess extraction and include an ORAI3-positive control |
| Broad smear instead of sharp band | Incomplete solubilization of membrane-associated ORAI3 is possible | Optimize membrane extraction and compare with a positive control |
Experiment evidence
Western blot images showing ORAI3 band
A09399 · ORAI3
M09399 · ORAI3
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for ORAI3 Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for ORAI3 in Western blot, you can use adrenal gland lysate, which has high HPA expression.
Positive control: Adrenal gland (IHC candidate; verify WB)
Negative control: Suggested KO / knockdown lysate
Loading controls: Run GAPDH, β-actin, and a total-protein stain such as Ponceau alongside the samples.
⚠️Feasibility: No tissue is reported as not detected, so use siRNA knockdown or a KO line for a clear negative control.
HPA tissue expression evidence for ORAI3
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 |
|---|---|---|---|---|
| Adrenal gland | glandular cells | High | Protein (IHC) | HPA → |
| Bone marrow | hematopoietic cells | High | Protein (IHC) | HPA → |
| Caudate | glial cells | High | Protein (IHC) | HPA → |
| Cerebellum | cells in molecular layer | High | Protein (IHC) | HPA → |
| Cerebral cortex | glial cells | High | Protein (IHC) | HPA → |
Lower expression tissues · IHC evidence, not confirmed WB-negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Duodenum | glandular cells | Low | Protein (IHC) | HPA → |
| Liver | cholangiocytes | Low | Protein (IHC) | HPA → |
| Adipose tissue | adipocytes | Medium | Protein (IHC) | HPA → |
| Appendix | glandular cells | Medium | Protein (IHC) | HPA → |
| Breast | adipocytes | Medium | Protein (IHC) | HPA → |
Section 3
Advanced ORAI3 Western Blot Tips
Deeper troubleshooting and optimisation questions for ORAI3, answered from its protein features.
Where should the ORAI3 monomer band appear?
Band shift · ORAI3 has a predicted mass of 31.5 kDa. No observed Western-blot band position is supplied, so use 31.5 kDa as a calculated reference, not a confirmed apparent mass.
Could alternative ORAI3 isoforms explain multiple bands?
Isoforms · The supplied record lists one isoform and no alternative sequence. It does not support assigning additional bands to alternative ORAI3 isoforms.
Can phosphorylation explain an ORAI3 band shift?
PTM · UniProt lists phosphoserine at positions 42 and 45, using UniProt sequence numbering. Check whether your antibody or paper uses the same numbering. These sites alone do not establish a visible shift or explain a difference from 31.5 kDa.
Does store depletion induce ORAI3 expression?
Induction · The supplied location note says ORAI3 colocalizes with STIM1 upon store depletion. It does not say ORAI3 abundance increases. Treat store depletion as a condition relevant to localization, not an established way to increase the ORAI3 Western-blot signal.
What transfer method to use for ORAI3 Western blot?
Transfer · ORAI3 is a multi-pass cell-membrane protein with a predicted mass of 31.5 kDa. Check transfer efficiency around that mass when optimizing a membrane-protein transfer. The supplied features do not specify a transfer method or settings.
How should blocking be optimized?
Blocking · Standard workflow guidance: follow the A09399 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 ORAI3 bands be quantified?
Quantitation · Compare bands from samples with consistent membrane-protein preparation and loading, because ORAI3 is located at the cell membrane. Report the band region used for quantitation; the record supplies no observed band position or evidence that a shifted band represents more ORAI3.
Do higher ORAI3 bands prove oligomerization?
Interpretation · ORAI3 participates in homomeric and heteromeric ORAI complexes, and native CRAC channels are most likely hexameric heteromers. That makes complexes relevant when investigating higher bands, but band position alone cannot establish their identity or subunit composition.
Boster reagents
ORAI3 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-Protein orai-3 ORAI3 Antibody
Cat # A09399
Real WB data
Anti-ORAI3 Monoclonal Antibody [2H2G9]
Cat # M09399
Two the supplier anti-ORAI3 antibodies have Western blot images: A09399 tested on A20 cell lysate at 1–4 μg/mL, and M09399 tested on rat spleen lysate at 1 μg/mL. These examples do not establish performance across all listed species or sample types.
Which to pick: Choose A09399 if mouse reactivity is needed; it lists human, mouse and rat and shows an A20 lysate blot. M09399 lists human and rat and shows a rat spleen blot. For rat spleen samples, M09399 has the directly matching image.
Source: BosterBio ORAI3 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry Q9BRQ5.
- Human Protein Atlas. ORAI3 tissue expression.
- PMC8150283 — target-verified WB comparison
- PMC8700618 — target-verified WB comparison
- PMC12583688 — target-verified WB comparison