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- Table of Contents
ISCU / Iron-sulfur cluster assembly enzyme ISCU · Western blot design guide
Design a Western Blot for ISCU
Real validated ISCU Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-ISCU 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
ISCU 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 | ~18 kDa | |
| Observed band | ~15 kDa | |
| Gel | 12% (catalog A05447-1) | |
| Positive control | Adrenal gland (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 | 2 isoform(s) |
Section 1
Real Curated ISCU Western Blot Protocols
The A05447-1 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | human Hela, human COLO320, human A549 (catalog A05447-1) |
| Gel % | 12% (catalog A05447-1) |
| Load | 30 ug; reducing conditions (catalog A05447-1) |
| Transfer | a nitrocellulose membrane at 150 mA for 50-90 minutes (catalog A05447-1) |
| Membrane | nitrocellulose membrane (catalog A05447-1) |
| Blocking | 5% non-fat milk/TBS for 1.5 hour at RT (catalog A05447-1) |
| Primary antibody | A05447-1 · 1:1000 (catalog A05447-1) |
| Primary incubation | overnight at 4°C (catalog A05447-1) |
| Secondary antibody | goat anti-rabbit IgG-HRP, 1:5000 (catalog A05447-1) |
| Secondary incubation | 1.5 hour at RT (catalog A05447-1) |
| Wash | TBS-0.1%Tween 3 times with 5 minutes each (catalog A05447-1) |
| Detection | ECL (catalog A05447-1) |
Section 2
What Is the Expected ISCU Western Blot Band Size?
ISCU is predicted at 18 kDa, but the supplied blot shows ~15 kDa; the cause of the difference is not established.
| Band at ~15 kDa | Empirical ISCU band; its difference from the 18 kDa predicted mass is unexplained |
| Band near 18 kDa | Consistent with the predicted sequence mass, pending identity confirmation |
| Band near 36 kDa | Could reflect retained isoform 1 homodimer; confirm its identity |
| Two bands in the monomer region | Could reflect isoforms 1 and 2 if they migrate differently; distinct bands are not established |
💡Expected ISCU appearanceISCU has a predicted sequence mass of 18 kDa, while the supplied Western blot shows ~15 kDa; the cause of this difference is unknown, so confirm band identity with an independent antibody or ISCU depletion.
| Predicted sequence mass | 18 kDa by sequence; the empirical band is ~15 kDa for an undetermined reason |
| Splice isoform 1 | Its individual mass and migration relative to isoform 2 are unspecified |
| Splice isoform 2 | Its individual mass and migration relative to isoform 1 are unspecified |
| Tyr-35-mediated isoform 1 homodimer | Could yield a band near twice the monomer mass if the dimer survives sample preparation and electrophoresis |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | Mitochondrial isoform 1 may be poorly represented in the sampled lysate | Check loading and compare a mitochondrial fraction |
| Band higher than expected | An isoform 1 homodimer may persist during electrophoresis | Compare denaturation conditions and confirm band identity by ISCU depletion |
| Band lower than expected | The observed ~15 kDa band differs from the 18 kDa prediction for an unknown reason | Compare with the supplied blot and confirm identity using ISCU depletion or an independent antibody |
| Multiple bands | Isoforms 1 and 2 may differ in migration, though distinct bands are unproven | Check isoform expression and confirm each band with isoform-aware controls |
| Weak or no signal | The mitochondrial signal may be diluted in a whole-cell sample | Check loading and antibody performance, then compare a mitochondrial fraction |
Experiment evidence
Western blot images showing ISCU band
A05447-1 · ISCU
A05447 · ISCU
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for ISCU Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for ISCU in Western blot, you can use adrenal gland lysate, which HPA scores as high expression.
Positive control: Adrenal gland (IHC candidate; verify WB)
Negative control: Adipose tissue (IHC candidate; verify WB)
Loading controls: Run GAPDH, β-actin, and a total-protein stain such as Ponceau alongside the samples.
⚠️Feasibility: HPA reports adipose tissue as not detected; ISCU's mitochondrial location may favor mitochondria-enriched lysate.
HPA tissue expression evidence for ISCU
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 → |
| Appendix | glandular cells | High | Protein (IHC) | HPA → |
| Caudate | glial cells | High | Protein (IHC) | HPA → |
| Cerebellum | Purkinje cells | High | Protein (IHC) | HPA → |
| Colon | endothelial cells | High | 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 → |
| Bone marrow | hematopoietic cells | Low | Protein (IHC) | HPA → |
| Spleen | cells in red pulp | Low | Protein (IHC) | HPA → |
| Breast | glandular cells | Medium | Protein (IHC) | HPA → |
| Bronchus | respiratory epithelial cells | Medium | Protein (IHC) | HPA → |
Section 3
Advanced ISCU Western Blot Tips
Deeper troubleshooting and optimisation questions for ISCU, answered from its protein features.
How should ISCU 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 could ISCU isoforms affect band interpretation?
Isoforms · Isoform 2 replaces residues 1–38 of the canonical sequence with a shorter sequence. It could therefore migrate differently from isoform 1. Check whether the antibody recognizes both N-terminal sequences before assigning separate bands to isoforms.
Which phosphorylation site should be considered when analyzing ISCU?
PTM · UniProt lists phosphoserine at position 14, modified by MTOR. State that this is a UniProt coordinate if comparing it with antibody or paper numbering. A phosphorylation annotation alone does not establish a visible band shift; use a site-specific assay to assess it.
Does this guide establish induction of ISCU?
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.
How should transfer be checked for ISCU?
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 A05447-1 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 can ISCU bands be quantified consistently?
Quantitation · Define the band or bands being measured before comparing samples. The observed band is approximately 15 kDa, and two isoforms are listed with different N-terminal sequences. Verify antibody recognition and band identity so that changes in one band are not mistaken for changes in total ISCU.
Why might ISCU appear near 15 kDa instead of 18 kDa?
Interpretation · The predicted mass is 18 kDa, while the observed band is approximately 15 kDa. ISCU has a transit-peptide keyword and two isoforms, but the supplied features do not establish which, if either, explains the difference. Use a molecular weight marker and validate band identity before assigning it to processed ISCU.
UniProt lists cysteine persulfides at positions 69 and 138. These are UniProt coordinates and may differ from numbering used elsewhere. Their presence does not establish a detectable mobility shift on a Western blot.
Isoform 1 is annotated in mitochondria. A mitochondrial-enriched sample may help assess that isoform alongside a whole-cell sample. The supplied location annotation does not establish where isoform 2 resides.
Isoform 1 is annotated as a homodimer, with Tyr-35 involved in dimerization. A higher Western-blot band alone does not establish that it is a dimer. Check antibody specificity and whether the band persists under denaturing conditions before assigning its identity.
UniProt lists cysteine persulfides at positions 69 and 138. These are UniProt coordinates and may differ from numbering used elsewhere. Their presence does not establish a detectable mobility shift on a Western blot.
Isoform 1 is annotated in mitochondria. A mitochondrial-enriched sample may help assess that isoform alongside a whole-cell sample. The supplied location annotation does not establish where isoform 2 resides.
Isoform 1 is annotated as a homodimer, with Tyr-35 involved in dimerization. A higher Western-blot band alone does not establish that it is a dimer. Check antibody specificity and whether the band persists under denaturing conditions before assigning its identity.
Boster reagents
ISCU 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-ISCU Antibody
Cat # A05447-1
Real WB data
Anti-ISCU Antibody
Cat # A05447
Two the supplier anti-ISCU antibodies, A05447-1 and A05447, list human, mouse, and rat reactivity and have WB images. A05447-1 documents specific lysates and an approximately 15 kDa band versus an expected 18 kDa; A05447’s caption provides fewer validation details.
Which to pick: Choose A05447-1 if you want documented WB examples in human cell lines and rat and mouse tissues, with a reported 1:1000 dilution. A05447 also has a WB image, but its caption identifies only extracts from various cell lines.
Source: BosterBio ISCU gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry Q9H1K1.
- Human Protein Atlas. ISCU tissue expression.
- PMC11902946 — target-verified WB comparison
- PMC12181301 — target-verified WB comparison
- PMC5875801 — target-verified WB comparison