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- Table of Contents
PRF1 / Perforin-1 · Western blot design guide
Design a Western Blot for PRF1
Real validated PRF1 Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-PRF1 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
PRF1 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 | ~61.4 kDa | |
| Observed band | ~61 kDa | |
| Gel | 10% (catalog PB9155) | |
| 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 | Glycosylated + Cleaved | |
| Caveat | Modification-state controls | |
| Gene-set association | MSigDB Hallmark membership | |
| Isoform | 1 isoform(s) |
Section 1
Real Curated PRF1 Western Blot Protocols
The PB9155 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | human HepG2, human HEL, human Daudi (catalog PB9155) |
| Gel % | 10% (catalog PB9155) |
| Load | 30 ug; reducing conditions (catalog PB9155) |
| Transfer | a nitrocellulose membrane at 150 mA for 50-90 minutes (catalog PB9155) |
| Membrane | nitrocellulose membrane (catalog PB9155) |
| Blocking | 5% non-fat milk/TBS for 1.5 hour at RT (catalog PB9155) |
| Primary antibody | PB9155 · 0.5 μg/mL (catalog PB9155) |
| Primary incubation | overnight at 4°C (catalog PB9155) |
| Secondary antibody | goat anti-rabbit IgG-HRP, 1:5000 (catalog PB9155) |
| Secondary incubation | 1.5 hour at RT (catalog PB9155) |
| Wash | TBS-0.1%Tween 3 times with 5 minutes each (catalog PB9155) |
| Detection | ECL (catalog PB9155) |
Section 2
What Is the Expected PRF1 Western Blot Band Size?
PRF1 has a predicted precursor mass of 61.4 kDa and an observed band at ~61 kDa; the cause of their small difference is not established.
| Band at ~61 kDa | Matches the empirical PRF1 band; confirm identity with an appropriate control |
| Band above ~61 kDa | Could reflect occupancy of the N-linked glycosylation sites; a visible shift is unproven |
| Band below ~61 kDa | Could reflect signal-peptide cleavage; the mature band size is not established |
| Broad band or smear | Could reflect variable occupancy of the two N-linked glycosylation sites |
| Little or no band in whole-cell lysate | PRF1 is secreted and also localizes to cytolytic granules |
💡Expected PRF1 appearanceUniProt predicts a 61.4 kDa precursor, while antibody blots show ~61 kDa; signal-peptide cleavage and N-linked glycosylation may affect migration, but the small difference has no established cause, so confirm band identity with controls.
| UniProt predicted mass | Sets a 61.4 kDa precursor baseline; the observed band is ~61 kDa |
| N-linked glycosylation at Asn205 | May raise apparent mass if occupied; its individual shift is unknown |
| N-linked glycosylation at Asn549 | May raise apparent mass if occupied; its individual shift is unknown |
| Signal peptide at residues 1–21 | Cleavage may lower mature-chain mass relative to the precursor; migration is unmeasured |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | Secreted PRF1 or PRF1 concentrated in cytolytic granules may be poorly represented | Check a suitable positive-control lysate and compare cell-associated and released material |
| Band higher than expected | Occupancy of Asn205 or Asn549 may alter migration | Compare matched samples before and after N-glycan removal and verify band identity |
| Band lower than expected | Signal-peptide cleavage could reduce mass, but the mature band size is unknown | Compare with a positive control and verify antibody recognition of the band |
| Broad smear instead of sharp band | Variable N-linked glycosylation is possible but unproven | Compare matched samples before and after N-glycan removal |
| Multiple bands | Different glycosylation or signal-peptide processing states are possible | Compare with a positive control and test N-glycan removal |
| Weak or no signal | Secretion or granule localization may limit PRF1 in the tested fraction | Test a suitable positive control and compare sample fractions |
Experiment evidence
Western blot images showing PRF1 band
PB9155 · PRF1
PB9155 · PRF1
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for PRF1 Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for PRF1 in Western blot, you can use spleen tissue, the strongest HPA positive candidate.
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 alongside the samples.
⚠️Feasibility: PRF1 is granule localized and secreted, so signal in whole-tissue lysate may vary.
HPA tissue expression evidence for PRF1
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 → |
| Endometrium | cells in endometrial stroma | Medium | Protein (IHC) | HPA → |
| Esophagus | squamous epithelial cells | Medium | Protein (IHC) | HPA → |
| Lymph node | non-germinal center cells | Medium | Protein (IHC) | HPA → |
| Oral mucosa | 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 → |
| Adrenal gland | glandular cells | Not detected | Protein (IHC) | HPA → |
| Appendix | glandular cells | Not detected | Protein (IHC) | HPA → |
| Bone marrow | hematopoietic cells | Not detected | Protein (IHC) | HPA → |
| Breast | adipocytes | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced PRF1 Western Blot Tips
Deeper troubleshooting and optimisation questions for PRF1, answered from its protein features.
How should PRF1 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 PRF1 isoforms explain multiple bands?
Isoforms · Only one isoform is supplied, with no alternative sequence. These features provide no basis for assigning additional bands to PRF1 isoforms.
Which PRF1 modifications matter when interpreting migration?
PTM · UniProt lists N-linked glycosylation at Asn205 and Asn549, using the supplied UniProt numbering, and nine disulfide bonds. These features may affect migration, but their presence alone does not establish a visible band shift.
Does this guide establish induction of PRF1?
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 PRF1 Western blot?
Transfer · These features do not specify a transfer method. PRF1 is found as a soluble monomer and in membrane-associated pore-forming oligomers. Check transfer recovery for the form and sample fraction being measured.
How should blocking be optimized?
Blocking · Standard workflow guidance: follow the PB9155 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 PRF1 be quantified across sample fractions?
Quantitation · PRF1 is reported in cytolytic granules, endosome lumen, secreted material, and cell membranes. Compare the same fraction and sample preparation across conditions; a change in one fraction alone may reflect redistribution rather than a change in total PRF1.
Does the observed ~61 kDa band match predicted PRF1 mass?
Interpretation · The observed ~61 kDa band is close to the 61.4 kDa predicted mass. PRF1 has a signal peptide at residues 1–21 and two N-linked glycosylation sites, but these features alone do not establish a visible shift or explain the apparent mass.
PRF1 homooligomerizes to form pore-forming rings and has nine disulfide bonds. Those features warrant checking whether sample preparation changes the band pattern. They do not, by themselves, identify a higher band as an oligomer.
The annotated signal peptide spans residues 1–21. Its removal changes the sequence present in mature PRF1, but the supplied features do not establish a separate visible band or its apparent mass.
PRF1 homooligomerizes to form pore-forming rings and has nine disulfide bonds. Those features warrant checking whether sample preparation changes the band pattern. They do not, by themselves, identify a higher band as an oligomer.
The annotated signal peptide spans residues 1–21. Its removal changes the sequence present in mature PRF1, but the supplied features do not establish a separate visible band or its apparent mass.
Boster reagents
PRF1 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-Perforin/PRF1 Antibody Picoband®
Cat # PB9155
PB9155 is a human-reactive anti-PRF1 antibody with a supplied Western blot image. Its caption reports an approximately 61 kDa band in human HepG2, HEL, Daudi, and HUH7 whole-cell lysates under reducing conditions. The supplied evidence covers these tested samples only.
Which to pick: PB9155 is the only listed option. Choose it for a human-sample WB design when a reported blot is useful; its image uses four human cell-line lysates. Confirm performance in your own sample and conditions.
Source: BosterBio PRF1 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry P14222.
- Human Protein Atlas. PRF1 tissue expression.
- PMC9929778 — target-verified WB comparison
- PMC10400057 — target-verified WB comparison