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- Table of Contents
SLC40A1 / Ferroportin · Western blot design guide
Design a Western Blot for SLC40A1
Real validated SLC40A1 Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-SLC40A1 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
SLC40A1 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 | ~62.5 kDa | |
| Observed band | ~70 kDa | |
| Gel | 5–20% (catalog A01953-2) | |
| Positive control | Bone marrow (IHC candidate; verify WB) | |
| Negative control | Adipose tissue (IHC candidate; verify WB) |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Glycosylated + Ubl conjugation | |
| Caveat | Modification-state controls | |
| Gene-set association | MSigDB C7 membership | |
| Isoform | 1 isoform(s) |
Section 1
Real Curated SLC40A1 Western Blot Protocols
The A01953-2 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | human placenta, rat liver, mouse spleen (catalog A01953-2) |
| Gel % | 5–20% (catalog A01953-2) |
| Load | 30 ug (catalog A01953-2) |
| Transfer | a nitrocellulose membrane at 150 mA for 50-90 minutes (catalog A01953-2) |
| Membrane | nitrocellulose membrane (catalog A01953-2) |
| Blocking | 5% non-fat milk/TBS for 1.5 hour at RT (catalog A01953-2) |
| Primary antibody | A01953-2 · 0.5 μg/mL (catalog A01953-2) |
| Primary incubation | overnight at 4°C (catalog A01953-2) |
| Secondary antibody | goat anti-rabbit IgG-HRP, 1:5000 (catalog A01953-2) |
| Secondary incubation | 1.5 hour at RT (catalog A01953-2) |
| Wash | TBS-0.1%Tween 3 times with 5 minutes each (catalog A01953-2) |
| Detection | ECL (catalog A01953-2) |
Section 2
What Is the Expected SLC40A1 Western Blot Band Size?
SLC40A1 is predicted at 62.5 kDa and observed at approximately 70 kDa; the cause of the difference is not established.
| Band at approximately 70 kDa | Empirical SLC40A1 band in reducing tissue lysates; confirm identity with controls |
| Band near 62.5 kDa | Near the predicted full-length polypeptide mass; identity still requires controls |
| Band above 62.5 kDa | Asn434 N-linked glycosylation could affect migration, but a visible shift is unproven |
| Weak band in soluble lysate fraction | Multi-pass membrane SLC40A1 may remain in the membrane fraction |
💡Expected SLC40A1 appearanceSLC40A1 has a predicted mass of 62.5 kDa and an empirical band at approximately 70 kDa; the cause of the difference is unestablished, so confirm band identity with appropriate controls.
| Predicted full-length mass | Places the unmodified sequence at 62.5 kDa |
| 571-residue sequence | Underlies the predicted 62.5 kDa polypeptide mass |
| Asn434 N-linked glycosylation site | Could increase apparent mass if occupied; no shift is established |
| Occupancy of the Asn434 site | Could affect migration, but occupancy and its effect are not supplied |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | Multi-pass membrane SLC40A1 may be poorly recovered | Check a membrane-enriched fraction and extraction conditions |
| Band higher than expected | The approximately 70 kDa empirical band exceeds the 62.5 kDa prediction; the cause is unestablished | Compare with a positive control and confirm identity by SLC40A1 depletion |
| Band lower than expected | A lower band has no established explanation in the supplied features | Confirm identity with SLC40A1 depletion or a second antibody |
| Multiple bands | Additional band identities are unestablished; only one isoform is listed | Compare bands with a positive control and SLC40A1 depletion |
| Weak or no signal | Membrane extraction may yield little SLC40A1 | Check membrane recovery and run a positive control |
Experiment evidence
Western blot images showing SLC40A1 band
A01953-2 · SLC40A1
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for SLC40A1 Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for SLC40A1 in Western blot, you can use bone marrow tissue.
Positive control: Bone marrow (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: As a multi-pass membrane protein, SLC40A1 may be easier to detect in a membrane-enriched fraction.
HPA tissue expression evidence for SLC40A1
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 |
|---|---|---|---|---|
| Bone marrow | hematopoietic cells | Medium | 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 → |
| Adrenal gland | glandular cells | Not detected | Protein (IHC) | HPA → |
| Appendix | glandular cells | Not detected | Protein (IHC) | HPA → |
| Breast | adipocytes | Not detected | Protein (IHC) | HPA → |
| Bronchus | respiratory epithelial cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced SLC40A1 Western Blot Tips
Deeper troubleshooting and optimisation questions for SLC40A1, answered from its protein features.
How should SLC40A1 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.
Are separate bands expected from annotated ferroportin isoforms?
Isoforms · The supplied UniProt record lists one isoform and no alternative sequence. It therefore provides no basis to assign additional bands to annotated isoforms. Evaluate band identity before interpreting extra bands.
Can glycosylation produce more than one ferroportin band?
PTM · UniProt lists one N-linked glycosylation site, Asn434 in its sequence numbering. Different glycosylation states could be considered when assessing multiple bands, but the site alone does not establish that distinct bands will appear. Compare treated and untreated samples if testing a glycosylation explanation.
Does this guide establish induction of SLC40A1?
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 SLC40A1 Western blot?
Transfer · UniProt identifies ferroportin as a multi-pass cell-membrane protein; the supplied apparent band is ~70 kDa. These features do not specify an optimal transfer method. Check transfer efficiency around 70 kDa and optimize the chosen method empirically.
How should blocking be optimized?
Blocking · Standard workflow guidance: follow the A01953-2 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 ferroportin bands be quantified?
Quantitation · Quantify the consistently identified ferroportin band near the supplied ~70 kDa apparent position across samples. If multiple bands appear, establish their identity before combining their signals. The annotated Asn434 glycosylation site alone does not show that every band represents ferroportin.
Why might ferroportin run near 70 kDa instead of 62.5 kDa?
Interpretation · The supplied apparent band is ~70 kDa, versus a predicted mass of 62.5 kDa. UniProt lists N-linked glycosylation at Asn434 and identifies ferroportin as a multi-pass membrane protein. These features are relevant to interpreting migration, but they do not establish the cause or size of the difference. Confirm band identity with an appropriate control.
UniProt lists an interaction with HAMP and states that iron increases HAMP affinity for ferroportin. That interaction does not, by itself, predict the direction or size of a Western-blot signal change after treatment. Compare treated samples with matched controls and measure the band directly.
Use the predicted 62.5 kDa mass and supplied ~70 kDa apparent band as reference points. UniProt lists one isoform, Asn434 N-linked glycosylation, and a multi-pass membrane location. None of these features alone identifies an unexpected band. Verify its identity with an appropriate control before assigning a cause.
UniProt lists an interaction with HAMP and states that iron increases HAMP affinity for ferroportin. That interaction does not, by itself, predict the direction or size of a Western-blot signal change after treatment. Compare treated samples with matched controls and measure the band directly.
Use the predicted 62.5 kDa mass and supplied ~70 kDa apparent band as reference points. UniProt lists one isoform, Asn434 N-linked glycosylation, and a multi-pass membrane location. None of these features alone identifies an unexpected band. Verify its identity with an appropriate control before assigning a cause.
Boster reagents
SLC40A1 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-SLC40A1 Antibody Picoband®
Cat # A01953-2
The catalog reports A01953-2, a rabbit polyclonal anti-SLC40A1 antibody with a Western blot image. The caption reports an approximately 70 kDa band (expected 63 kDa) in human placenta, rat liver, and mouse spleen and liver lysates. Evidence is limited to these tested samples and conditions.
Which to pick: A01953-2 is the only listed option. Its stated reactivity is human, mouse, and rat; its WB image includes samples from each species. For a starting protocol, the caption reports 30 µg lysate per lane and 0.5 µg/mL primary antibody.
Source: BosterBio SLC40A1 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry Q9NP59.
- Human Protein Atlas. SLC40A1 tissue expression.
- PMC7347119 — target-verified WB comparison
- PMC12910788 — target-verified WB comparison
- PMC12972206 — target-verified WB comparison