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- Table of Contents
ANKRD54 / Ankyrin repeat domain-containing protein 54 · Western blot design guide
Design a Western Blot for ANKRD54
Source-linked ANKRD54 Western blot protocol options, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-ANKRD54 WB antibodies. Everything you need to plan the experiment before you commit precious samples.
Evidence assembled October 2026 · For research use; verify linked source records and product datasheet before use
ANKRD54 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 | ~32.5 kDa | |
| Observed band | 68 kDa | |
| Gel | 12–15% (standard starting point) | |
| Positive control | Gallbladder (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 | Blocking peptide control | |
| Gene-set association | MSigDB C7 membership | |
| Isoform | 2 isoform(s) |
Section 1
Source-Linked ANKRD54 Western Blot Protocol Options
The A13999 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | mouse kidney tissue lysate (catalog A13999) |
| 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 | Western blot analysis of LIAR in mouse kidney tissue lysate with LIAR antibody at 1 μg/mL in (A) the absence and (B) the presence of blocking peptide (catalog A13999) |
| Primary antibody | A13999 · 1 μg/mL (catalog A13999) |
| 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 ANKRD54 Western Blot Band Size?
ANKRD54 is predicted at 32.5 kDa, while an empirical band is reported at 68 kDa; the cause of the difference is not established.
| Band near 68 kDa | Empirical antibody-associated band; its identity and reason for migrating above the predicted mass require confirmation |
| Band near 32.5 kDa | Consistent with the UniProt predicted mass of ANKRD54 |
| Additional bands at different positions | Could represent isoforms 1 and 2, whose masses and migration have not been established |
| Closely spaced bands | Could reflect phosphorylation at Ser58 or Ser63, though a visible shift has not been demonstrated |
💡Expected ANKRD54 appearanceANKRD54 has a predicted mass of 32.5 kDa and an empirical antibody-associated band at 68 kDa; the difference is unexplained, so confirm band identity with blocking peptide and independent controls.
| UniProt predicted mass | Predicts 32.5 kDa; the empirical band is 68 kDa, with no established cause for the difference |
| Isoforms 1 and 2 | May differ in size, but their individual masses and migration are unknown |
| N-acetylalanine at residue 2 | Adds an N-terminal modification without an established visible band shift |
| Phosphoserine at residue 58 | May affect migration, but no visible shift is established |
| Phosphoserine at residue 63 | May affect migration, but no visible shift is established |
| Situation | Likely cause | Next action |
|---|---|---|
| Band higher than expected | The reported 68 kDa band exceeds the 32.5 kDa prediction for an unknown reason | Check blocking peptide competition and confirm identity with knockdown or an independent antibody |
| Band lower than expected | An isoform is possible, but neither isoform has a supplied mass | Confirm ANKRD54 identity with knockdown or isoform-specific expression |
| Multiple bands | Isoforms 1 and 2 or phosphorylation at Ser58 and Ser63 are possible, but distinct bands are unproven | Compare knockdown samples and test phosphatase treatment if needed |
| Weak or no signal | ANKRD54 distribution between nucleus and cytoplasm changes during the cell cycle | Check nuclear and cytoplasmic fractions and include a positive-control lysate |
| No band in lysate | Nuclear accumulation may limit recovery from a cytoplasm-enriched preparation | Check whole-cell and nuclear lysates alongside a positive control |
Experiment evidence
Western blot images showing ANKRD54 band
A13999 · ANKRD54
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for ANKRD54 Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for ANKRD54 in Western blot, you can use gallbladder tissue, which has high HPA expression.
Positive control: Gallbladder (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 clean negative control.
HPA tissue expression evidence for ANKRD54
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 |
|---|---|---|---|---|
| Gallbladder | glandular cells | High | Protein (IHC) | HPA → |
| Pancreas | exocrine glandular cells | High | Protein (IHC) | HPA → |
| Testis | sertoli cells | High | Protein (IHC) | HPA → |
| Thyroid gland | glandular cells | High | Protein (IHC) | HPA → |
| Adrenal gland | glandular cells | Medium | Protein (IHC) | HPA → |
Lower expression tissues · IHC evidence, not confirmed WB-negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adipose tissue | adipocytes | Low | Protein (IHC) | HPA → |
| Bone marrow | hematopoietic cells | Low | Protein (IHC) | HPA → |
| Caudate | neuronal cells | Low | Protein (IHC) | HPA → |
| Heart muscle | cardiomyocytes | Low | Protein (IHC) | HPA → |
| Hippocampus | neuronal cells | Low | Protein (IHC) | HPA → |
Section 3
Advanced ANKRD54 Western Blot Tips
Deeper troubleshooting and optimisation questions for ANKRD54, answered from its protein features.
How should ANKRD54 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.
How might ANKRD54 isoforms affect band detection?
Isoforms · UniProt lists isoforms 1 and 2. Isoform 2 lacks canonical residues 1–139 and replaces residues 140–198 with a different sequence. Check whether the antibody epitope is present in each isoform before interpreting missing or additional bands.
Which phosphorylation sites matter when interpreting ANKRD54 bands?
PTM · UniProt lists phosphoserine at positions 58 and 63, using its supplied sequence coordinates. These sites provide a reason to consider phosphorylation, but their presence alone does not establish a visible band shift.
UniProt lists N-acetylalanine at position 2 in its supplied coordinates. This modification alone does not demonstrate a visible band shift or explain the reported 68 kDa band.
UniProt lists N-acetylalanine at position 2 in its supplied coordinates. This modification alone does not demonstrate a visible band shift or explain the reported 68 kDa band.
Should EPO stimulation change the ANKRD54 signal?
Induction · The supplied location note says EPO stimulation induces nuclear accumulation by similarity. Compare nuclear and cytoplasmic fractions if testing this response. That annotation does not establish a change in total ANKRD54 abundance.
How should transfer be checked for ANKRD54?
Transfer · Standard workflow guidance: verify transfer efficiency for the intended target size before interpreting a weak signal. Use total-protein assessment and optimize transfer for the membrane, gel and apparatus; the labelled catalog values take precedence.
How should blocking be optimized?
Blocking · Standard workflow guidance: follow the A13999 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 ANKRD54 be quantified across cell-cycle samples?
Quantitation · ANKRD54 shuttles between nucleus and cytoplasm during the cell cycle and is also listed at the midbody. Keep the sampled fraction consistent and interpret changes in a fraction's signal in light of this localization.
Does the 68 kDa band match ANKRD54's predicted mass?
Interpretation · The supplied predicted mass is 32.5 kDa, while the reported apparent band is 68 kDa. The listed features do not establish why they differ. Do not identify a 68 kDa band as ANKRD54 from size alone.
Consider the two listed isoforms and whether the antibody recognizes their distinct sequences. The supplied features also list phosphorylation at positions 58 and 63, but do not establish that it produces separate bands. Band position alone cannot assign an unexpected band to an isoform or modification.
Consider the two listed isoforms and whether the antibody recognizes their distinct sequences. The supplied features also list phosphorylation at positions 58 and 63, but do not establish that it produces separate bands. Band position alone cannot assign an unexpected band to an isoform or modification.
Boster reagents
ANKRD54 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-LIAR ANKRD54 Antibody
Cat # A13999
The catalog reports A13999 for ANKRD54 Western blotting, with reported Human, Mouse, and Rat reactivity. Its WB image shows mouse kidney lysate tested at 1 μg/mL with and without blocking peptide; the supplied evidence does not show other sample contexts.
Which to pick: A13999 is the only listed option. Its WB image documents mouse kidney lysate at 1 μg/mL, including a blocking-peptide comparison. For other samples, the listed reactivity alone does not establish WB performance.
Source: BosterBio ANKRD54 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry Q6NXT1.
- Human Protein Atlas. ANKRD54 tissue expression.
- PMC5378395 — target-verified WB comparison