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- Table of Contents
MFF / Mitochondrial fission factor · Western blot design guide
Design a Western Blot for MFF
Real validated MFF Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-MFF 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
MFF 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 | ~38.5 kDa | |
| Observed band | ~26 kDa | |
| Gel | 5–20% (catalog A02563-1) | |
| Positive control | Appendix (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 | Observed below predicted | |
| Gene-set association | MSigDB C7 membership | |
| Isoform | 5 isoform(s) |
Section 1
Real Curated MFF Western Blot Protocols
The A02563-1 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | human Hela, human HepG2, human SiHa (catalog A02563-1) |
| Gel % | 5–20% (catalog A02563-1) |
| Load | 30 ug; reducing conditions (catalog A02563-1) |
| Transfer | a nitrocellulose membrane at 150 mA for 50-90 minutes (catalog A02563-1) |
| Membrane | nitrocellulose membrane (catalog A02563-1) |
| Blocking | 5% non-fat milk/TBS for 1.5 hour at RT (catalog A02563-1) |
| Primary antibody | A02563-1 · 0.5 μg/mL (catalog A02563-1) |
| Primary incubation | overnight at 4°C (catalog A02563-1) |
| Secondary antibody | goat anti-rabbit IgG-HRP, 1:5000 (catalog A02563-1) |
| Secondary incubation | 1.5 hour at RT (catalog A02563-1) |
| Wash | TBS-0.1%Tween 3 times with 5 minutes each (catalog A02563-1) |
| Detection | ECL (catalog A02563-1) |
Section 2
What Is the Expected MFF Western Blot Band Size?
MFF is predicted at 38.5 kDa, while antibody QC detects approximately 26 kDa; the cause of this difference is not established.
| Band near 26 kDa | Empirical MFF band in reducing whole-cell lysates; confirm identity with controls |
| Band near 38.5 kDa | Near the predicted sequence mass; identity requires confirmation |
| Band near twice the monomer size | Possible retained MFF homodimer if the preparation preserves it |
| Several bands at different positions | May reflect MFF isoforms 1–5, whose migration differences are unestablished |
💡Expected MFF appearanceUniProt predicts 38.5 kDa, while antibody QC reports an MFF band near 26 kDa in reducing lysates; the difference is unexplained, so confirm band identity with knockdown or an independent antibody.
| Predicted sequence mass | 38.5 kDa by UniProt; the empirical band is near 26 kDa, with no established cause for the difference |
| MFF homodimer | Could appear near twice the monomer mass if preserved during preparation |
| Isoform 1 | Its individual mass and migration are not supplied |
| Isoforms 2, 3, 4 and 5 | Alternative sequences may change apparent size, but their masses and separation are not supplied |
| Situation | Likely cause | Next action |
|---|---|---|
| Band lower than expected | The reported 26 kDa band is below the 38.5 kDa prediction; its cause is unestablished | Confirm identity by MFF knockdown or an independent antibody |
| Band higher than expected | A retained MFF homodimer is possible | Compare fully denatured and reduced samples |
| Multiple bands | MFF has five named isoforms, but distinct band positions are unestablished | Compare isoform expression and check band identity by knockdown |
| No band in lysate | Membrane-associated MFF may be poorly recovered or solubilized | Check membrane extraction and compare an enriched mitochondrial fraction |
| Weak or no signal | Low recovery of mitochondrial outer-membrane MFF is possible | Check membrane-protein recovery and use a positive-control lysate |
Experiment evidence
Western blot images showing MFF band
A02563-1 · MFF
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for MFF Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for MFF in Western blot, you can use appendix tissue, which has medium HPA staining.
Positive control: Appendix (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: MFF is an outer-membrane protein, so mitochondrial-enriched lysate may improve detection.
HPA tissue expression evidence for MFF
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 |
|---|---|---|---|---|
| Appendix | glandular cells | Medium | Protein (IHC) | HPA → |
| Cerebellum | Purkinje cells | Medium | Protein (IHC) | HPA → |
| Cerebral cortex | neuronal cells | Medium | Protein (IHC) | HPA → |
| Colon | peripheral nerve/ganglion | Medium | Protein (IHC) | HPA → |
| Esophagus | squamous 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 → |
| Caudate | glial cells | Not detected | Protein (IHC) | HPA → |
| Heart muscle | cardiomyocytes | Not detected | Protein (IHC) | HPA → |
| Liver | cholangiocytes | Not detected | Protein (IHC) | HPA → |
| Ovary | follicle cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced MFF Western Blot Tips
Deeper troubleshooting and optimisation questions for MFF, answered from its protein features.
How should MFF 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.
Which MFF isoforms could produce smaller bands?
Isoforms · Isoforms 2–5 lack canonical residues 1–26. They also lack residues 174–198, 199–271, 174–271 and 174–251, respectively. These deletions make smaller products plausible, but the supplied features do not assign a specific isoform to the observed 26 kDa band.
Check whether the antibody’s target lies in a region retained by the isoforms you want to measure. Canonical residues 1–26 are absent from isoforms 2–5; additional regions within 174–271 are absent from particular isoforms. An antibody targeting a missing region may fail to detect that isoform.
Check whether the antibody’s target lies in a region retained by the isoforms you want to measure. Canonical residues 1–26 are absent from isoforms 2–5; additional regions within 174–271 are absent from particular isoforms. An antibody targeting a missing region may fail to detect that isoform.
Could phosphorylation explain an MFF band shift?
PTM · UniProt lists phosphothreonine at canonical residues 115 and 200, phosphoserine at 155, 157, 172, 202, 229 and 233, and multiple phosphorylation labels at 295. Keep these as UniProt canonical coordinates when comparing antibody or paper numbering. Site annotations alone do not demonstrate a visible shift or explain the 26 kDa band.
Does this guide establish induction of MFF?
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 MFF Western blot?
Transfer · MFF is annotated as a single-pass outer-membrane protein with a predicted canonical mass of 38.5 kDa. The supplied features do not specify a transfer method. Check transfer efficiency around both 38.5 kDa and the reported 26 kDa band when assessing your setup.
How should blocking be optimized?
Blocking · Standard workflow guidance: follow the A02563-1 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 MFF bands be quantified?
Quantitation · Specify which band you measured and use the same band definition across samples. MFF has five isoforms with different deletions, and the reported 26 kDa band differs from the canonical predicted mass. Verify that your antibody recognizes the isoforms you intend to quantify before combining signals from different bands.
Why might MFF appear near 26 kDa instead of 38.5 kDa?
Interpretation · The canonical predicted mass is 38.5 kDa, while the reported apparent band is about 26 kDa. MFF has five isoforms with sequence deletions that could yield smaller products. These features alone do not identify the 26 kDa band or establish why it migrates there; confirm its identity with an antibody targeting a retained region.
Compare band sizes with the five annotated isoforms and check whether the antibody target is retained in each. MFF is also annotated as a phosphoprotein and homodimer, but those features alone cannot identify an unexpected band. The reported 26 kDa band should not be assigned to an isoform or modification from apparent mass alone.
Compare band sizes with the five annotated isoforms and check whether the antibody target is retained in each. MFF is also annotated as a phosphoprotein and homodimer, but those features alone cannot identify an unexpected band. The reported 26 kDa band should not be assigned to an isoform or modification from apparent mass alone.
Boster reagents
MFF 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-MFF Antibody Picoband®
Cat # A02563-1
The catalog reports one anti-MFF antibody, A02563-1, with stated human, mouse, and rat reactivity. Its Western blot image shows an approximately 26 kDa band in the named human cell lines and rat and mouse brain lysates. No publication evidence is supplied.
Which to pick: A02563-1 is the only listed option. Its WB image includes human HeLa, HepG2, SiHa, MOLT-4, and HEL cell lysates, plus rat and mouse brain lysates, tested at 30 µg per lane with antibody at 0.5 µg/mL.
Source: BosterBio MFF gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry Q9GZY8.
- Human Protein Atlas. MFF tissue expression.
- PMC5627902 — target-verified WB comparison
- PMC5341724 — target-verified WB comparison
- PMC4397917 — target-verified WB comparison