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- Table of Contents
FECH / Ferrochelatase, mitochondrial · Western blot design guide
Design a Western Blot for FECH
Source-linked FECH Western blot protocol options, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-FECH 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
FECH 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 | ~47.9 kDa | |
| Observed band | ~48 kDa | |
| Gel | 10% (catalog A05156-2) | |
| Positive control | Kidney (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 | Acetylated | |
| Caveat | Modification-state controls | |
| Gene-set association | MSigDB Hallmark membership | |
| Isoform | 2 isoform(s) |
Section 1
Source-Linked FECH Western Blot Protocol Options
The A05156-2 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | human K562, human HEL, human RT4, human A549, rat heart, rat kidney, mouse heart, mouse kidney (catalog A05156-2) |
| Gel % | 10% (catalog A05156-2) |
| Load | 30 ug; reducing conditions (catalog A05156-2) |
| Transfer | a nitrocellulose membrane at 150 mA for 50-90 minutes (catalog A05156-2) |
| Membrane | nitrocellulose membrane (catalog A05156-2) |
| Blocking | 5% non-fat milk/TBS for 1.5 hour at RT (catalog A05156-2) |
| Primary antibody | A05156-2 · 1:1000 (catalog A05156-2) |
| Primary incubation | overnight at 4°C (catalog A05156-2) |
| Secondary antibody | goat anti-rabbit IgG-HRP, 1:5000 (catalog A05156-2) |
| Secondary incubation | 1.5 hour at RT (catalog A05156-2) |
| Wash | TBS-0.1%Tween 3 times with 5 minutes each (catalog A05156-2) |
| Detection | ECL (catalog A05156-2) |
Section 2
What Is the Expected FECH Western Blot Band Size?
FECH is 47.9 kDa predicted and ~48 kDa observed; no biological cause for the small difference is established.
| Band at ~48 kDa | matches the observed FECH band and its 47.9 kDa predicted mass |
| Band near ~96 kDa | could reflect a retained FECH homodimer |
| Band near ~192 kDa | could reflect a retained FECH homotetramer |
| Additional bands at different sizes | could reflect isoforms 1 and 2; distinct migration is unconfirmed |
💡Expected FECH appearanceFECH has a predicted mass of 47.9 kDa and an observed band at ~48 kDa; confirm band identity with appropriate antibody and sample controls.
| Predicted FECH mass | 47.9 kDa predicted; the observed band is ~48 kDa |
| FECH homodimer | could appear near twice the monomer size if the complex survives electrophoresis |
| Isoform 1 | may migrate differently from isoform 2; its mass is not supplied |
| Isoform 2 | may migrate differently from isoform 1; its mass is not supplied |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | FECH is associated with the mitochondrial inner membrane and may be underrepresented in the sample | check mitochondrial protein recovery and enrich a mitochondrial fraction |
| Band higher than expected | a FECH homodimer or homotetramer may remain associated | compare fully denatured samples and verify band identity |
| Band lower than expected | an alternative isoform is possible, but its mass is unknown | compare isoform-specific controls and verify band identity |
| Multiple bands | isoforms or retained FECH oligomers are possible | compare denaturation conditions and isoform-specific controls |
| Fragments below expected size | FECH may have degraded during sample preparation | prepare fresh samples with protease inhibitors and verify fragment identity |
Experiment evidence
Western blot images showing FECH band
A05156-2 · FECH
A05156 · FECH
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for FECH Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for FECH in Western blot, you can use kidney tissue, which shows high HPA expression.
Positive control: Kidney (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 FECH.
⚠️Feasibility: HPA supports a clear tissue contrast, though mitochondrial enrichment may improve FECH detection.
HPA tissue expression evidence for FECH
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 |
|---|---|---|---|---|
| Kidney | cells in tubules | High | Protein (IHC) | HPA → |
| Prostate | glandular cells | High | Protein (IHC) | HPA → |
| Adrenal gland | glandular cells | Medium | Protein (IHC) | HPA → |
| Appendix | glandular cells | Medium | Protein (IHC) | HPA → |
| Bronchus | respiratory epithelial 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 → |
| Smooth muscle | smooth muscle cells | Not detected | Protein (IHC) | HPA → |
| Soft tissue | fibroblasts | Not detected | Protein (IHC) | HPA → |
| Bone marrow | hematopoietic cells | Low | Protein (IHC) | HPA → |
| Breast | glandular cells | Low | Protein (IHC) | HPA → |
Section 3
Advanced FECH Western Blot Tips
Deeper troubleshooting and optimisation questions for FECH, answered from its protein features.
How should FECH 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 FECH isoforms produce separate bands?
Isoforms · UniProt lists isoforms 1 and 2. In isoform 2, residue 64 changes from K to KRYESNI, adding six residues. This may change calculated mass slightly, but the feature does not establish that two bands will resolve on a blot.
Which FECH modification sites matter when interpreting bands?
PTM · In the supplied UniProt coordinates, K57 and K415 are acetylation sites, while K138 and K415 are succinylation sites. The two modifications at K415 are alternate. These annotations do not establish a detectable mobility shift; check which isoform and numbering convention an antibody or paper uses.
Does this guide establish induction of FECH?
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 FECH Western blot?
Transfer · No transfer method is specified by the supplied features. FECH is a peripheral protein on the matrix side of the mitochondrial inner membrane. Check recovery and transfer of the band near 48 kDa in your preparation, and optimize the method empirically.
How should blocking be optimized?
Blocking · Standard workflow guidance: follow the A05156-2 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 FECH bands be quantified?
Quantitation · Quantify the band near 48 kDa consistently across samples. FECH is located at the mitochondrial inner membrane, so keep sample preparation consistent when comparing signal. If additional bands appear, do not combine them with the 48 kDa band without establishing their identity.
Does FECH’s observed band match its predicted mass?
Interpretation · The reported band near 48 kDa matches the predicted 47.9 kDa. The listed modifications and isoforms alone do not establish a visible shift or explain any small difference in apparent mass.
UniProt reports FECH homodimers and homotetramers. A higher band could prompt investigation of oligomers, but those annotations do not show that either assembly survives Western blot preparation. Confirm band identity before assigning it to an FECH complex.
UniProt reports FECH homodimers and homotetramers. A higher band could prompt investigation of oligomers, but those annotations do not show that either assembly survives Western blot preparation. Confirm band identity before assigning it to an FECH complex.
Boster reagents
FECH 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-FECH antibody
Cat # A05156-2
Real WB data
Anti-Ferrochelatase, mitochondrial FECH Antibody
Cat # A05156
Both the supplier anti-FECH antibodies, A05156-2 and A05156, list human, mouse, and rat reactivity and have Western blot images. A05156-2 shows an approximately 48 kDa band in named human cell and rat/mouse tissue lysates. A05156's caption does not name its cell lines.
Which to pick: For the documented samples, consider A05156-2: its image includes K562, HEL, RT4, A549, and rat and mouse heart and kidney lysates. A05156 also has a WB image, but its cell lines are unnamed. Both captions report a 1:1000 primary antibody dilution.
Source: BosterBio FECH gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry P22830.
- Human Protein Atlas. FECH tissue expression.
- PMC7726024 — target-verified WB comparison
- PMC11994340 — target-verified WB comparison
- PMC5452042 — target-verified WB comparison