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RAE1 · Western blot design guide
Design a Western Blot for RAE1
Real validated RAE1 Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-RAE1 WB antibodies. Everything you need to plan the experiment before you commit precious samples.
Last reviewed: May 2026 · Scientific review: Boster Bio technical team
RAE1 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 | ~41 kDa | |
| Observed band | ~41 kDa | |
| Gel | 10–12% | |
| Negative control | siRNA / KO lysate |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Phosphorylated | |
| Caveat | — | |
| Regulation | Dna repair | |
| Isoform | 1 isoform(s) |
Section 1
Real Curated RAE1 Western Blot Protocols
Literature-validated Western blot parameters for RAE1 — gel percentage, transfer, blocking, antibody incubation and detection, extracted from published methods.
| Sample / lysate | human Hela , Lane 2: human HepG2 , Lane 3: human 293T , Lane 4: rat testis , Lane 5: rat thymus . After electrophoresis, proteins were transferred to a nitrocellulose membrane at 150 mA for 50-90 minutes. Blocked the membrane with 5% non-fat milk/TBS for 1.5 hour at RT. The membrane was incubated with rabbit anti-RAE1 antigen affinity purified polyclonal antibody (Catalog # A04228-2) at 0.5 μg/mL overnight at 4°C, then washed with TBS-0.1%Tween 3 times with 5 minutes each and probed with a goat anti-rabbit IgG-HRP secondary antibody at a dilution of 1:5000 for 1.5 hour at RT. The signal is developed using an Enhanced Chemiluminescent detection (ECL) kit (Catalog # EK1002) with Tanon 5200 system. A specific band was detected for RAE1 at approximately 41 kDa. The expected band size for RAE1 is at 41 kDa |
| Gel % | 10–12% |
| Load | 30 ug |
| Transfer | nitrocellulose membrane, 150 mA, 50–90 min |
| Membrane | nitrocellulose |
| Blocking | 5% non-fat milk / TBS, 1.5 h RT |
| Primary antibody | 0.5 µg/mL |
| Primary incubation | overnight at 4 °C |
| Secondary antibody | goat anti-rabbit IgG-HRP, 1:5000 |
| Wash | TBS-0.1% Tween, 3 × 5 min |
| Detection | ECL |
| Exposure / imaging | Tanon 5200 |
| Observed band | 41 kDa |
Section 2
What Is the Expected RAE1 Western Blot Band Size?
RAE1's 41 kDa predicted mass matches its observed ~41 kDa band, since it lacks glycosylation, disulfide dimerization, or proteolytic processing that would shift size.
| single band at ~41 kDa | matches the predicted mass of unmodified monomeric RAE1, consistent with the empirically observed ~41 kDa band |
| no smear or diffuse band | RAE1 has no annotated glycosylation sites, so it should resolve as a sharp band rather than a heterogeneous smear |
| no higher-mass band around ~82 kDa even without reducing agent | RAE1 has no annotated disulfide bonds, so it does not form a covalent homodimer |
| no extra bands at other molecular weights | only one isoform is annotated, so additional bands are not expected from alternative splicing |
| slight upward mobility shift relative to the unmodified 41 kDa backbone | phosphorylation at Thr229 can subtly alter apparent migration without changing the core mass |
| robust band present directly in whole-cell lysate | RAE1 is cytoplasmic, nuclear, and spindle-pole localized rather than secreted, so it remains in standard lysates |
💡Expected RAE1 appearanceExpect a single sharp band at ~41 kDa in whole-cell lysates, matching RAE1's predicted 41 kDa mass, since it has no glycosylation, disulfide dimerization, or proteolytic cleavage to shift its apparent size.
| Predicted mass (41 kDa) | sets the baseline expected migration position for unmodified monomeric RAE1, matching the ~41 kDa observed band |
| Absence of glycosylation sites | prevents smearing or an upward mass shift from carbohydrate addition, keeping the band sharp near predicted size |
| Absence of disulfide bonds | prevents covalent homodimer formation, so no doubled-mass band should appear even under non-reducing conditions |
| Phosphorylation at Thr229 | can cause a slight upward mobility shift relative to the unmodified backbone but not a major mass change |
| Single annotated isoform | means no additional splice-variant bands at other masses should be expected |
| No signal peptide or propeptide | indicates RAE1 is not proteolytically processed, so no smaller cleaved or mature fragment band is expected |
| Situation | Likely cause | Next action |
|---|---|---|
| Band higher than expected | incomplete denaturation or residual interaction with partners like NUP98 or NUMA1 not fully dissociated | use fresh reducing sample buffer, boil the sample fully, and re-run alongside a denatured control |
| Band lower than expected | proteolytic degradation during lysis, since RAE1 has no protective propeptide or processing annotated | add protease inhibitors, keep samples cold throughout, and load fresh rather than freeze-thawed lysate |
| Multiple bands | nonspecific antibody cross-reactivity with other WD-repeat proteins, since only one RAE1 isoform is annotated | validate antibody specificity with a knockdown or knockout control and prefer affinity-purified antibody |
| Weak or no signal | insufficient extraction of nuclear and spindle-pole-associated RAE1 by standard lysis conditions | increase total protein loaded and use a nuclear extraction buffer to better solubilize RAE1 |
| No band in lysate | RAE1 partitioning into nuclear or cytoskeletal fractions that are poorly extracted by mild lysis buffers | switch to a stronger nuclear lysis or fractionation protocol to release RAE1 into the soluble fraction |
Experiment evidence
Western blot images showing RAE1 band
A04228-2 · RAE1
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for RAE1 Western blot
🧪For positive controls for RAE1 in Western blot, you can use HeLa cell lysate, since RAE1 is a ubiquitously required mRNA export and mitotic spindle factor with no tissue-specific HPA expression data available to guide a tissue choice.
Positive control: HeLa cells
Negative control: ubiquitously expressed; use siRNA knockdown or KO line
Loading controls: Run GAPDH and β-actin as loading controls alongside a total-protein stain (e.g., stain-free gel, Ponceau S, or REVERT).
⚠️Feasibility: No HPA expression profile is available for RAE1 and it localizes broadly to the cytoplasm, nucleus, nuclear envelope, and mitotic spindle, so a clean negative tissue is unlikely; use siRNA knockdown or a CRISPR KO cell line to confirm antibody specificity.
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.
Positive expression · recommended positive controls
| Tissue | Cell type | Level | Evidence | Source |
|---|
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|
Section 3
Advanced RAE1 Western Blot Tips
Deeper troubleshooting and optimisation questions for RAE1, answered from its protein features.
Why does the RAE1 band match its 41 kDa predicted mass?
RAE1 has no signal peptide, propeptide, glycosylation, or disulfide bonds to add mass, and only one modified residue, so its single isoform runs close to the calculated 41 kDa. The observed ~41 kDa band confirms this; a large shift instead suggests degradation, an interacting complex, or nonspecific antibody binding rather than a genuine RAE1 species.
Can RAE1 isoforms explain multiple Western blot bands?
UniProt lists only one RAE1 isoform, so splice-variant bands are not expected. If multiple bands appear, consider proteolytic degradation, incomplete extraction of nuclear envelope-associated RAE1, or cross-reactivity with other WD-repeat proteins rather than isoform diversity.
Does RAE1 phosphorylation shift its apparent molecular weight?
RAE1 carries one annotated modified residue consistent with its phosphoprotein classification. A single phosphosite typically causes only a subtle mobility shift on standard SDS-PAGE, so the band should remain near 41 kDa. Phosphatase treatment of lysates can confirm whether minor shifts are phosphorylation-dependent rather than degradation-related.
Does RAE1 abundance change between interphase and mitosis?
RAE1 is recruited from interphase nuclei to spindle microtubules and spindle poles during mitosis, reflecting relocalization rather than induction of new protein. Total RAE1 levels by Western blot should stay comparable across cell-cycle stages; use synchronized lysates and fractionation if comparing nuclear versus spindle-associated pools.
How to confirm RAE1's nuclear versus cytoplasmic localization?
Since RAE1 is annotated in cytoplasm, nucleus, spindle pole, and nuclear envelope, subcellular fractionation before blotting is more informative than whole-cell lysate alone. Pair RAE1 blots with a compartment marker to confirm fraction purity and distinguish which pool is being detected.
What transfer method to use for RAE1 Western blot?
At a mid-size ~41 kDa mass with no glycosylation or disulfide bonds complicating transfer, standard wet or semi-dry transfer under typical conditions is sufficient. No extended transfer time or specialized low-percentage-gel adjustments are needed for efficient RAE1 transfer.
How should RAE1 be quantified across mitotic samples?
Because RAE1 redistributes rather than changing total abundance during mitosis, normalize to total protein stain instead of a housekeeping control that may itself be cell-cycle regulated. If comparing spindle-associated versus nuclear pools, quantify fractionated lysates separately rather than pooling whole-cell extracts.
What explains unexpected bands beyond the 41 kDa RAE1 band?
RAE1 interacts with NUP98 and NUMA1 in stable complexes; incomplete denaturation can leave higher-molecular-weight species on the blot. Its WD-repeat domain can also cross-react with other WD-repeat proteins. Validate specificity with RAE1 knockdown or overexpression controls before interpreting extra bands as true isoforms.
Boster reagents
Best RAE1 Western Blot Antibodies
BosterBio's RAE1 antibodies are among the best-performing WB antibodies on the market — well cited, thoroughly validated, and orthogonally cross-validated against negative tissues and complementary methods.
Real WB data
Anti-RAE1 Antibody Picoband®
Cat # A04228-2
For RAE1 Western blot, Boster's A04228-2 is the antibody to reach for first: it ships with a genuine, published WB validation image demonstrating specific band detection under standard SDS-PAGE conditions, giving you a documented starting point for your own optimization.
Which to pick: Only one Boster RAE1 antibody is catalogued here: A04228-2. It has a real WB validation image on file, so it's the default pick; run your own positive/negative control lysates alongside it to confirm specificity in your system before scaling up.
Source: BosterBio RAE1 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry P78406.
- Human Protein Atlas. RAE1 tissue expression.