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- Table of Contents
ALYREF / THO complex subunit 4 · Western blot design guide
Design a Western Blot for ALYREF
Real validated ALYREF Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-ALYREF 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
ALYREF 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 | ~26.9 kDa | |
| Observed band | ~32 kDa | |
| Gel | 12% (catalog M03580) | |
| Positive control | Adipose tissue (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 + Acetylated | |
| Caveat | Modification-state controls | |
| Gene-set association | MSigDB Hallmark membership | |
| Isoform | 1 isoform(s) |
Section 1
Real Curated ALYREF Western Blot Protocols
The M03580 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | human 293T, human SH-SY5Y, human U251 (catalog M03580) |
| Gel % | 12% (catalog M03580) |
| Load | 30 ug; reducing conditions (catalog M03580) |
| Transfer | a nitrocellulose membrane at 150 mA for 50-90 minutes (catalog M03580) |
| Membrane | nitrocellulose membrane (catalog M03580) |
| Blocking | 5% non-fat milk/TBS for 1.5 hour at RT (catalog M03580) |
| Primary antibody | M03580 · 1:500 (catalog M03580) |
| Primary incubation | overnight at 4°C (catalog M03580) |
| Secondary antibody | goat anti-rabbit IgG-HRP, 1:5000 (catalog M03580) |
| Secondary incubation | 1.5 hour at RT (catalog M03580) |
| Wash | TBS-0.1%Tween 3 times with 5 minutes each (catalog M03580) |
| Detection | ECL (catalog M03580) |
Section 2
What Is the Expected ALYREF Western Blot Band Size?
ALYREF is predicted at 26.9 kDa but observed near 32 kDa in reducing whole-cell blots; the cause of the difference is not established.
| Band near 32 kDa | Empirical ALYREF band in reducing whole-cell lysates |
| Band near 27 kDa | Near the 26.9 kDa sequence-predicted mass; confirm its identity |
| Faint band in whole-cell lysate | ALYREF may be easier to detect in a nuclear fraction |
| Additional nearby bands | Could reflect modified ALYREF species; band identities require validation |
💡Expected ALYREF appearanceALYREF has a predicted mass of 26.9 kDa, while reducing whole-cell blots show an empirical band at approximately 32 kDa; the cause of the difference is unestablished, so confirm identity with appropriate controls.
| Predicted ALYREF mass | 26.9 kDa by sequence; the empirical band is approximately 32 kDa |
| Ser8 phosphorylation | Present as an annotated modification; its effect on migration is unestablished |
| Arg38 methylation | Alternative methylation states are annotated; their effect on migration is unestablished |
| Lys86 acetylation | Present as an annotated modification; its effect on migration is unestablished |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | Nuclear ALYREF may be poorly represented in the sampled lysate | Check a nuclear fraction and verify transfer and antibody performance |
| Band higher than expected | The observed 32 kDa band exceeds the 26.9 kDa prediction; the cause is unestablished | Compare with the empirical 32 kDa reference and confirm identity by ALYREF depletion |
| Band lower than expected | A smaller band has no assigned ALYREF form in the supplied evidence | Check sample integrity and confirm identity with ALYREF depletion |
| Multiple bands | Modified species or unrelated antibody binding are possible; band identities are unestablished | Use ALYREF depletion and a second antibody to identify specific bands |
| Weak or no signal | The sampled fraction may contain little nuclear ALYREF | Try a nuclear fraction and include a validated positive lysate |
Experiment evidence
Western blot images showing ALYREF band
M03580 · ALYREF
A03580-3 · ALYREF
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for ALYREF Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for ALYREF in Western blot, you can use adipose tissue lysate, which has high HPA expression.
Positive control: Adipose tissue (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 HPA tissue is listed as not detected, so use siRNA knockdown or a KO line as the negative control.
HPA tissue expression evidence for ALYREF
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 |
|---|---|---|---|---|
| Adipose tissue | Adipocytes | High | Protein (IHC) | HPA → |
| Adrenal gland | Glandular cells | High | Protein (IHC) | HPA → |
| Appendix | Glandular cells | High | Protein (IHC) | HPA → |
| Bone marrow | Hematopoietic cells | High | Protein (IHC) | HPA → |
| Breast | Adipocytes | High | Protein (IHC) | HPA → |
Lower expression tissues · IHC evidence, not confirmed WB-negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adipose tissue | Adipocytes | High | Protein (IHC) | HPA → |
| Adrenal gland | Glandular cells | High | Protein (IHC) | HPA → |
| Appendix | Glandular cells | High | Protein (IHC) | HPA → |
| Bone marrow | Hematopoietic cells | High | Protein (IHC) | HPA → |
| Breast | Adipocytes | High | Protein (IHC) | HPA → |
Section 3
Advanced ALYREF Western Blot Tips
Deeper troubleshooting and optimisation questions for ALYREF, answered from its protein features.
How should ALYREF 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 ALYREF isoforms explain multiple bands?
Isoforms · The supplied UniProt record lists one isoform and no alternative sequence. These features provide no basis for assigning additional bands to ALYREF isoforms; verify their identity before doing so.
Which phosphorylation sites matter when interpreting ALYREF bands?
PTM · UniProt lists phosphoserine at positions 8, 94, and 239, using UniProt sequence numbering. Compare those coordinates with your antibody's numbering convention. These sites establish possible phosphorylation states, but do not establish a visible band shift.
UniProt lists arginine methylation at positions 38, 58, 63, 71, 197, 204, and 220, with alternate annotations at 38, 197, and 204. It also lists citrulline at 141. All positions use UniProt numbering. These features alone cannot identify the cause of an extra band.
UniProt lists arginine methylation at positions 38, 58, 63, 71, 197, 204, and 220, with alternate annotations at 38, 197, and 204. It also lists citrulline at 141. All positions use UniProt numbering. These features alone cannot identify the cause of an extra band.
Does this guide establish induction of ALYREF?
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 ALYREF Western blot?
Transfer · ALYREF has a predicted mass of 26.9 kDa, and its reported apparent band is approximately 32 kDa. Choose transfer conditions that retain proteins in this size range, then check the membrane and post-transfer gel to assess transfer efficiency.
How should blocking be optimized?
Blocking · Standard workflow guidance: follow the M03580 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 ALYREF be quantified across cellular fractions?
Quantitation · UniProt places ALYREF in the nucleus, including nuclear speckles, and in the cytoplasm. Quantify the approximately 32 kDa band within each fraction and use appropriate fraction controls when comparing nuclear and cytoplasmic signal.
Why might ALYREF appear near 32 kDa instead of 26.9 kDa?
Interpretation · The reported apparent band is approximately 32 kDa, while the predicted mass is 26.9 kDa. UniProt lists several modifications, but their presence alone does not establish the cause of this difference. Compare the band with a molecular weight marker and confirm its identity independently.
UniProt describes ALYREF as a homomultimer, predominantly a hexamer when bound to the EJC-RNA complex. That association does not establish that a higher band is an ALYREF multimer on a Western blot. Verify band identity before assigning it to a complex or modification.
UniProt describes ALYREF as a homomultimer, predominantly a hexamer when bound to the EJC-RNA complex. That association does not establish that a higher band is an ALYREF multimer on a Western blot. Verify band identity before assigning it to a complex or modification.
Boster reagents
ALYREF 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-Aly Rabbit Monoclonal Antibody
Cat # M03580
Real WB data
Anti-ALYREF Antibody Picoband®
Cat # A03580-3
The catalog reports two anti-ALYREF antibodies for Western blot, M03580 and A03580-3, both reported reactive with human, mouse, and rat. Their product blots show bands around 32 and 35 kDa, respectively, above the expected 27 kDa. Evidence is limited to the supplied product blots.
Which to pick: Both have WB images. Choose M03580 for a monoclonal tested on human, rat, and mouse cell lysates. Choose A03580-3 for a polyclonal tested on cell and tissue lysates from those species. Match the documented specimen and conditions to your experiment.
Source: BosterBio ALYREF gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry Q86V81.
- Human Protein Atlas. ALYREF tissue expression.
- PMC10942997 — target-verified WB comparison
- PMC12171766 — target-verified WB comparison
- PMC7802773 — target-verified WB comparison