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PPIF / Peptidyl-prolyl cis-trans isomerase F, mitochondrial · Western blot design guide
Design a Western Blot for PPIF
Source-linked PPIF Western blot protocol options, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-PPIF 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
PPIF 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 | ~22 kDa | |
| Observed band | ~18 kDa | |
| Gel | 5–20% (catalog M02803) | |
| Negative control | Suggested KO / knockdown lysate |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Acetylated | |
| Caveat | Band-identity controls | |
| Gene-set association | MSigDB Hallmark membership | |
| Isoform | 2 isoform(s) |
Section 1
Source-Linked PPIF Western Blot Protocol Options
The M02803 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | human Hela, human HEL, human PC-3, human HepG2, rat PC-12, rat C6, mouse RAW264.7, mouse Neuro-2a (catalog M02803) |
| Gel % | 5–20% (catalog M02803) |
| Load | 30 ug; reducing conditions (catalog M02803) |
| Transfer | a nitrocellulose membrane at 150 mA for 50-90 minutes (catalog M02803) |
| Membrane | nitrocellulose membrane (catalog M02803) |
| Blocking | 5% non-fat milk/TBS for 1.5 hour at RT (catalog M02803) |
| Primary antibody | M02803 · 1:500 (catalog M02803) |
| Primary incubation | overnight at 4°C (catalog M02803) |
| Secondary antibody | goat anti-rabbit IgG-HRP, 1:5000 (catalog M02803) |
| Secondary incubation | 1.5 hour at RT (catalog M02803) |
| Wash | TBS-0.1%Tween 3 times with 5 minutes each (catalog M02803) |
| Detection | ECL (catalog M02803) |
Section 2
What Is the Expected PPIF Western Blot Band Size?
PPIF is predicted at 22 kDa but observed near 18 kDa in antibody QC; the cause of the difference is not established.
| Band near 18 kDa | Empirical PPIF band in reducing whole-cell blots; its difference from the 22 kDa prediction is unexplained |
| Band near 22 kDa | Matches the predicted PPIF sequence mass; confirm identity experimentally |
| Additional discrete bands | Could reflect isoforms 1 and 2, whose separate migration has not been established |
| Faint band in whole-cell lysate | PPIF is located in the mitochondrial matrix, so signal may depend on mitochondrial content |
💡Expected PPIF appearanceUniProt predicts 22 kDa, while antibody QC shows a band near 18 kDa in reducing whole-cell lysates; the cause of the difference is unconfirmed, so verify band identity with appropriate controls.
| Predicted PPIF mass | 22 kDa from sequence, compared with the empirical band near 18 kDa; the difference is unexplained |
| Isoform 1 | Named splice isoform; its individual mass and migration are unspecified |
| Isoform 2 | Named splice isoform; its size relative to isoform 1 is unspecified |
| Mitochondrial transit peptide | Processing could lower mass, but cleavage and its contribution to the 18 kDa band are unconfirmed |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | Mitochondrial matrix PPIF may be scarce in the loaded whole-cell sample | Check loading and compare with a mitochondria-enriched fraction |
| Band lower than expected | The observed 18 kDa band is below the 22 kDa prediction for an undetermined reason | Confirm identity with PPIF knockdown or another validated antibody |
| Multiple bands | Isoforms 1 and 2 are annotated, but distinct band sizes are unverified | Test band identity with PPIF depletion and isoform-specific evidence |
| Weak or no signal | Low mitochondrial content or insufficient PPIF signal in the sample | Verify transfer and loading, then compare mitochondrial enrichment |
| Fragments below expected size | Sample proteolysis could produce smaller immunoreactive bands | Prepare fresh lysate with protease inhibitors and confirm bands by PPIF depletion |
Experiment evidence
Western blot images showing PPIF band
M02803 · PPIF
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for PPIF Western blot
🧪For positive controls for PPIF in Western blot, you can use a PPIF-verified mitochondria-enriched lysate, since no HPA-positive tissue or cell is supplied.
Positive control: No high/medium HPA tissue identified
Negative control: Suggested KO / knockdown lysate
Loading controls: Run GAPDH, β-actin, and a total-protein stain such as Ponceau alongside the samples.
⚠️Feasibility: With no HPA expression data, tissue controls cannot be identified; verify a positive sample and use knockdown or KO for a negative control.
HPA tissue expression evidence for PPIF
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 |
|---|---|---|---|---|
| No high/medium HPA tissues identified in the supplied evidence. | ||||
Lower expression tissues · IHC evidence, not confirmed WB-negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| No lower-expression tissue rows available in the supplied evidence. | ||||
Section 3
Advanced PPIF Western Blot Tips
Deeper troubleshooting and optimisation questions for PPIF, answered from its protein features.
How should PPIF 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 PPIF isoforms produce different bands?
Isoforms · Yes. UniProt lists isoforms 1 and 2. In isoform 2, canonical residues 139–207 are replaced by WMASMLCSVTSKRAWTS. This substantial sequence change could affect migration. Check which isoform your antibody recognizes before assigning a second band.
Which PPIF modification sites matter when interpreting bands?
PTM · UniProt lists lysine 67 as alternatively acetylated or succinylated; lysines 73 and 197 as benzoylated; lysines 86, 175, and 190 as succinylated; lysine 167 as acetylated; and cysteine 203 as S-nitrosylated. These are UniProt coordinates; antibody or paper numbering may differ.
No. The annotated acetylation, succinylation, benzoylation, and S-nitrosylation sites identify possible modified residues. Their presence alone does not establish that a modification causes a resolvable shift or explains the ~18 kDa band.
The supplied annotation says inorganic phosphate increases PPIF association with mitochondrial ATP synthase, while cyclosporin A decreases it; both claims are marked by similarity. It does not say either treatment changes PPIF abundance or predicts a Western blot band shift.
No. The annotated acetylation, succinylation, benzoylation, and S-nitrosylation sites identify possible modified residues. Their presence alone does not establish that a modification causes a resolvable shift or explains the ~18 kDa band.
The supplied annotation says inorganic phosphate increases PPIF association with mitochondrial ATP synthase, while cyclosporin A decreases it; both claims are marked by similarity. It does not say either treatment changes PPIF abundance or predicts a Western blot band shift.
Does this guide establish induction of PPIF?
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 PPIF?
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 M02803 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 PPIF when multiple bands appear?
Quantitation · Quantify the same defined band across samples and state whether it is the ~18 kDa band or another species. Because PPIF has two annotated isoforms and several modification sites, do not combine bands as total PPIF without evidence that each band is PPIF and that the antibody detects them comparably.
Why is the observed PPIF band ~18 kDa instead of 22 kDa?
Interpretation · The supplied predicted mass is 22 kDa, while the observed band is ~18 kDa. PPIF is mitochondrial and carries a transit peptide keyword, so processing is worth considering, but no cleavage position is supplied. These features do not establish the cause of the mass difference.
PPIF associates with mitochondrial ATP synthase according to the supplied annotation. That association may inform follow-up experiments, but it does not establish that a higher Western blot band is a PPIF complex. Verify the identity of any unexpected band before assigning it.
PPIF associates with mitochondrial ATP synthase according to the supplied annotation. That association may inform follow-up experiments, but it does not establish that a higher Western blot band is a PPIF complex. Verify the identity of any unexpected band before assigning it.
Boster reagents
PPIF 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-Cyclophilin F Rabbit Monoclonal Antibody
Cat # M02803
The catalog reports one anti-PPIF antibody, M02803, a rabbit monoclonal with a Western blot image using human, rat, and mouse cell lysates. The caption reports a band near 18 kDa, versus 22 kDa expected; independent specificity validation is not provided.
Which to pick: M02803 is the only listed option. Its Western blot caption documents selected human, rat, and mouse cell lysates under reducing conditions, using 30 µg per lane and a 1:500 primary antibody dilution. Check whether those samples and conditions fit your experiment.
Source: BosterBio PPIF gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry P30405.
- Human Protein Atlas. PPIF tissue expression.
- PMC12552154 — target-verified WB comparison
- PMC13050221 — target-verified WB comparison
- PMC2710407 — target-verified WB comparison