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- Table of Contents
NFS1 / Cysteine desulfurase · Western blot design guide
Design a Western Blot for NFS1
Source-linked NFS1 Western blot protocol options, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-NFS1 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
NFS1 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 | ~50.2 kDa | |
| Observed band | Approximately 45 kDa | |
| Gel | 5–20% (catalog A05061-1) | |
| 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 | — | |
| Caveat | Band below predicted mass | |
| Gene-set association | MSigDB Hallmark membership | |
| Isoform | 3 isoform(s) |
Section 1
Source-Linked NFS1 Western Blot Protocol Options
The A05061-1 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | human Caco-2, human HEL, human HepG2, human SIHA, rat RH35, rat NRK, mouse HEPA1-6, mouse B16-F10 (catalog A05061-1) |
| Gel % | 5–20% (catalog A05061-1) |
| Load | 30 ug; reducing conditions (catalog A05061-1) |
| Transfer | a nitrocellulose membrane at 150 mA for 50-90 minutes (catalog A05061-1) |
| Membrane | nitrocellulose membrane (catalog A05061-1) |
| Blocking | 5% non-fat milk/TBS for 1.5 hour at RT (catalog A05061-1) |
| Primary antibody | A05061-1 · 0.5 μg/mL (catalog A05061-1) |
| Primary incubation | overnight at 4°C (catalog A05061-1) |
| Secondary antibody | goat anti-rabbit IgG-HRP, 1:5000 (catalog A05061-1) |
| Secondary incubation | 1.5 hour at RT (catalog A05061-1) |
| Wash | TBS-0.1%Tween 3 times with 5 minutes each (catalog A05061-1) |
| Detection | ECL (catalog A05061-1) |
Section 2
What Is the Expected NFS1 Western Blot Band Size?
NFS1 is predicted at 50.2 kDa, while antibody QC detects approximately 45 kDa; the cause of the difference is not established.
| Band near 45 kDa | Empirical NFS1 band in reducing whole-cell lysates; confirm identity with antibody controls |
| Band near 50.2 kDa | Near the predicted full-length mass; identity requires confirmation |
| Band near 100 kDa | Could reflect retained mitochondrial NFS1 homodimer if it survives electrophoresis |
| Multiple bands at different sizes | Could reflect NFS1 isoforms, though distinct migration is unproven |
💡Expected NFS1 appearanceNFS1 has a predicted mass of 50.2 kDa, while antibody QC shows a band at approximately 45 kDa in reducing whole-cell lysates; confirm band identity with ordinary antibody controls.
| Predicted NFS1 mass | 50.2 kDa for the supplied sequence; the empirical band is approximately 45 kDa |
| Mitochondrial isoform homodimer | Could appear near twice the monomer mass if the dimer survives electrophoresis |
| Mitochondrial isoform | Isoform-specific apparent mass is not supplied |
| Cytosolic isoform | Isoform-specific apparent mass is not supplied |
| Isoform 3 | Isoform-specific apparent mass is not supplied |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | Mitochondrial NFS1 may be dilute in whole-cell lysate | Check loading and examine a mitochondrial-enriched fraction |
| Band higher than expected | Mitochondrial NFS1 can form a homodimer | Compare denaturing and reducing sample preparation and verify band identity |
| Band lower than expected | Approximately 45 kDa is the empirical band, but its difference from 50.2 kDa is unexplained | Compare with the antibody QC band and verify specificity by NFS1 depletion |
| Multiple bands | NFS1 has Mitochondrial, Cytosolic, and 3 isoforms, but their migration is unspecified | Test band identity by NFS1 depletion and, if available, isoform-specific detection |
| Weak or no signal | The mitochondrial pool may be dilute in the tested sample | Check loading and antibody performance, then examine a mitochondrial-enriched fraction |
Experiment evidence
Western blot images showing NFS1 band
A05061-1 · NFS1
A05061-1 · NFS1
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for NFS1 Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for NFS1 in Western blot, you can use adrenal gland tissue, which shows 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 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 NFS1
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 → |
| Bronchus | respiratory epithelial cells | High | Protein (IHC) | HPA → |
| Cerebellum | GLUC cells - cytoplasm/membrane | High | Protein (IHC) | HPA → |
| Cerebral cortex | neuronal cells | High | Protein (IHC) | HPA → |
Lower expression tissues · IHC evidence, not confirmed WB-negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Bone marrow | hematopoietic cells | Low | Protein (IHC) | HPA → |
| Lymph node | germinal center cells | Low | Protein (IHC) | HPA → |
| Adipose tissue | adipocytes | Medium | Protein (IHC) | HPA → |
| Breast | glandular cells | Medium | Protein (IHC) | HPA → |
| Caudate | neuronal cells | Medium | Protein (IHC) | HPA → |
Section 3
Advanced NFS1 Western Blot Tips
Deeper troubleshooting and optimisation questions for NFS1, answered from its protein features.
What should I check when unexpected NFS1 bands appear?
Band shift · First compare band positions with the approximately 45 kDa observed band and the 50.2 kDa predicted mass. Then consider the three listed isoforms and whether the antibody epitope is retained in each. The listed modifications and isoform differences do not, by themselves, identify an unexpected band.
Which NFS1 isoforms could produce different bands?
Isoforms · UniProt lists Mitochondrial, Cytosolic, and isoform 3. Relative to the supplied sequence, Cytosolic lacks residues 1–60 and isoform 3 lacks residues 136–186. Check whether the antibody’s epitope falls within either missing region before interpreting band differences.
Do NFS1 modifications predict a visible band shift?
PTM · UniProt lists N6-(pyridoxal phosphate)lysine at position 258 and cysteine persulfide at position 381, using UniProt coordinates. Their presence does not establish a visible shift. Avoid assigning a shifted band to either modification without additional evidence.
Does this guide establish induction of NFS1?
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 NFS1?
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 A05061-1 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 should NFS1 bands be quantified across samples?
Quantitation · Use the same defined band or band set across samples, and state whether the measurement includes multiple isoforms. The Cytosolic and isoform 3 sequences omit different regions, so check antibody epitope coverage before treating their signals as comparable.
Why might NFS1 run near 45 kDa instead of 50.2 kDa?
Interpretation · The supplied apparent band is approximately 45 kDa, while the predicted mass is 50.2 kDa. NFS1 has alternative isoforms with missing sequence, but these features alone do not establish which isoform produced the band or explain the mass difference. Confirm band identity with an appropriate control.
UniProt places the Mitochondrial isoform in mitochondria and lists a Cytosolic isoform. Compare mitochondrial and cytosolic fractions when identifying bands, while checking the antibody epitope against the isoform sequence differences. Fraction location alone does not prove band identity.
UniProt places the Mitochondrial isoform in mitochondria and lists a Cytosolic isoform. Compare mitochondrial and cytosolic fractions when identifying bands, while checking the antibody epitope against the isoform sequence differences. Fraction location alone does not prove band identity.
Boster reagents
NFS1 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-NFS1 Antibody Picoband®
Cat # A05061-1
A05061-1 is listed for human, mouse, and rat NFS1. Its WB image uses whole-cell lysates from human, rat, and mouse cell lines and shows a band near 45 kDa, versus the expected 50 kDa. Evidence is limited to this example.
Which to pick: A05061-1 is the only listed option and has a WB image. Check whether your sample resembles the captioned cell lines, and account for the observed 45 kDa band relative to the stated 50 kDa expectation.
Source: BosterBio NFS1 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry Q9Y697.
- Human Protein Atlas. NFS1 tissue expression.
- PMC11876915 — target-verified WB comparison
- PMC5875801 — target-verified WB comparison
- PMC3027643 — target-verified WB comparison