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AIFM2 / Ferroptosis suppressor protein 1 · Western blot design guide
Design a Western Blot for AIFM2
Source-linked AIFM2 Western blot protocol options, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-AIFM2 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
AIFM2 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 | ~40.5 kDa | |
| Observed band | ~41 kDa | |
| Gel | 10% (catalog A06541-2) | |
| Positive control | Spleen (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 + Ubl conjugation | |
| Caveat | Modification-state controls | |
| Gene-set association | MSigDB C7 membership | |
| Isoform | 2 isoform(s) |
Section 1
Source-Linked AIFM2 Western Blot Protocol Options
The A06541-2 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | human HepG2, human A549, human Caco-2, human U251 (catalog A06541-2) |
| Gel % | 10% (catalog A06541-2) |
| Load | 30 ug; reducing conditions (catalog A06541-2) |
| Transfer | a nitrocellulose membrane at 150 mA for 50-90 minutes (catalog A06541-2) |
| Membrane | nitrocellulose membrane (catalog A06541-2) |
| Blocking | 5% non-fat milk/TBS for 1.5 hour at RT (catalog A06541-2) |
| Primary antibody | A06541-2 · 1:1000 (catalog A06541-2) |
| Primary incubation | overnight at 4°C (catalog A06541-2) |
| Secondary antibody | goat anti-rabbit IgG-HRP, 1:5000 (catalog A06541-2) |
| Secondary incubation | 1.5 hour at RT (catalog A06541-2) |
| Wash | TBS-0.1%Tween 3 times with 5 minutes each (catalog A06541-2) |
| Detection | ECL (catalog A06541-2) |
Section 2
What Is the Expected AIFM2 Western Blot Band Size?
AIFM2 is predicted at 40.5 kDa and observed near 41 kDa; the small difference has no established cause in the supplied evidence.
| Band near 41 kDa | Matches the empirical AIFM2 band and its 40.5 kDa predicted mass |
| Single band near 41 kDa | Consistent with AIFM2; its two named isoforms need not resolve separately |
| Additional band at another position | Could reflect an AIFM2 isoform, but its identity requires validation |
| Weak band in a soluble fraction | AIFM2 also localizes to membranes and lipid droplets |
💡Expected AIFM2 appearanceAIFM2 has a predicted mass of 40.5 kDa and an empirical band near 41 kDa in reducing whole-cell lysates; confirm band identity with an AIFM2 depletion or knockout control.
| UniProt predicted mass | 40.5 kDa predicted; the empirical band is near 41 kDa |
| Alternative splicing | May affect apparent size, but no isoform-specific mass or migration is supplied |
| Isoform 1 | Named isoform with no supplied individual band size |
| Isoform 2 | Named isoform with no supplied individual band size |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | AIFM2 may be poorly recovered from membrane-associated material | Check extraction and compare with a membrane-enriched fraction |
| Band higher than expected | Its identity is unestablished; no glycosylation or disulfide feature explains a shift | Compare with an AIFM2 depletion or knockout control |
| Band lower than expected | An isoform or fragment is possible, but its size is unspecified | Verify identity with AIFM2 depletion and an antibody to another epitope |
| Multiple bands | Two isoforms are named, but their migration is unknown | Check which bands respond to AIFM2 depletion |
| Weak or no signal | Recovery or abundance may vary across AIFM2 cellular compartments | Check lysate loading and compare appropriate subcellular fractions |
Experiment evidence
Western blot images showing AIFM2 band
A06541-2 · AIFM2
A06541 · AIFM2
A06541 · AIFM2
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for AIFM2 Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for AIFM2 in Western blot, you can use spleen lysate, supported by high HPA expression.
Positive control: Spleen (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: Membrane and lipid droplet localization may make thorough protein extraction important for reliable detection.
HPA tissue expression evidence for AIFM2
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 |
|---|---|---|---|---|
| Spleen | cells in red pulp | High | Protein (IHC) | HPA → |
| Bone marrow | hematopoietic cells | Medium | Protein (IHC) | HPA → |
| Lung | alveolar cells | Medium | Protein (IHC) | HPA → |
| Small intestine | glandular cells | Medium | Protein (IHC) | HPA → |
| Stomach | glandular 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 → |
| Appendix | glandular cells | Not detected | Protein (IHC) | HPA → |
| Breast | adipocytes | Not detected | Protein (IHC) | HPA → |
| Bronchus | respiratory epithelial cells | Not detected | Protein (IHC) | HPA → |
| Cerebellum | cells in granular layer | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced AIFM2 Western Blot Tips
Deeper troubleshooting and optimisation questions for AIFM2, answered from its protein features.
How should AIFM2 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 AIFM2 isoforms produce different bands?
Isoforms · Yes. UniProt lists two isoforms. Relative to isoform 1, isoform 2 lacks residues 99–138 and has SLLG in place of S at position 206, using canonical UniProt coordinates. These sequence differences could change migration, but they do not establish where either isoform will appear on a blot.
Which documented AIFM2 modification should I consider?
PTM · UniProt lists N6-acetyllysine at canonical residue 168, modified by KAT2B. Check whether an antibody recognizes a region affected by this modification when interpreting its signal. The feature alone does not establish a visible band shift.
Does this guide establish induction of AIFM2?
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 AIFM2?
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 A06541-2 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 I quantify AIFM2 across samples?
Quantitation · Keep the sampled fraction consistent because AIFM2 has several reported locations and may translocate under oxidative stress. If multiple bands appear, quantify each consistently and avoid treating a change in one fraction as proof of a change in total protein.
Why does AIFM2 appear near 41 kDa?
Interpretation · The observed band near 41 kDa is close to the predicted 40.5 kDa mass. The supplied features do not establish a cause for the small difference in apparent mass.
The supplied feature says interaction with KPNA2 and IPO5 likely mediates nuclear translocation upon oxidative stress. It supports checking nuclear and other fractions after stress, but does not establish increased total AIFM2 abundance.
UniProt places AIFM2 at lipid droplets, the cell membrane, the cytoplasm and the mitochondrial membrane; its keywords also include nucleus. Compare matched fractions when assessing distribution, and account for the amount of material loaded from each fraction.
First consider the two listed isoforms and whether the samples contain different cellular fractions. The documented acetylation at canonical residue 168 does not, by itself, explain an extra band. Verify band identity before assigning it to an isoform or modification.
The supplied feature says interaction with KPNA2 and IPO5 likely mediates nuclear translocation upon oxidative stress. It supports checking nuclear and other fractions after stress, but does not establish increased total AIFM2 abundance.
UniProt places AIFM2 at lipid droplets, the cell membrane, the cytoplasm and the mitochondrial membrane; its keywords also include nucleus. Compare matched fractions when assessing distribution, and account for the amount of material loaded from each fraction.
First consider the two listed isoforms and whether the samples contain different cellular fractions. The documented acetylation at canonical residue 168 does not, by itself, explain an extra band. Verify band identity before assigning it to an isoform or modification.
Boster reagents
AIFM2 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-FSP1/AIFM2 Antibody
Cat # A06541-2
Real WB data
Anti-AMID AIFM2 Antibody
Cat # A06541
Two the supplier anti-AIFM2 antibodies list Western blot images. A06541-2 has a detailed blot caption reporting a 41 kDa band in four human cell lysates. A06541 has a brief caption without identified samples or band size; neither caption establishes broad validation.
Which to pick: For the documented human cell lysate conditions, choose A06541-2. For monkey or mouse samples, A06541 lists those species as reactive, but its Western blot caption does not identify the tested cells. Both products list a Western blot image.
Source: BosterBio AIFM2 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry Q9BRQ8.
- Human Protein Atlas. AIFM2 tissue expression.
- PMC12674247 — target-verified WB comparison
- PMC10195139 — target-verified WB comparison
- PMC12826024 — target-verified WB comparison