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- Table of Contents
CENPF / Centromere protein F · Western blot design guide
Design a Western Blot for CENPF
Real validated CENPF Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-CENPF 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
CENPF 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 | ~357.5 kDa | |
| Observed band | ~358 kDa | |
| Gel | 5–20% (catalog A03311-1) | |
| 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 + Acetylated | |
| Caveat | Phosphorylation-state controls | |
| Gene-set association | MSigDB Hallmark membership | |
| Isoform | 1 isoform(s) |
Section 1
Real Curated CENPF Western Blot Protocols
The A03311-1 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | human Hela (catalog A03311-1) |
| Gel % | 5–20% (catalog A03311-1) |
| Load | 30 ug; reducing conditions (catalog A03311-1) |
| Transfer | a nitrocellulose membrane at 150 mA for 50-90 minutes (catalog A03311-1) |
| Membrane | nitrocellulose membrane (catalog A03311-1) |
| Blocking | 5% non-fat milk/TBS for 1.5 hour at RT (catalog A03311-1) |
| Primary antibody | A03311-1 · 0.5 μg/mL (catalog A03311-1) |
| Primary incubation | overnight at 4°C (catalog A03311-1) |
| Secondary antibody | goat anti-rabbit IgG-HRP, 1:5000 (catalog A03311-1) |
| Secondary incubation | 1.5 hour at RT (catalog A03311-1) |
| Wash | TBS-0.1%Tween 3 times with 5 minutes each (catalog A03311-1) |
| Detection | ECL (catalog A03311-1) |
Section 2
What Is the Expected CENPF Western Blot Band Size?
CENPF is predicted at 357.5 kDa and observed near 358 kDa; the small difference has no established biological cause.
| Band at approximately 358 kDa in whole-cell lysate | matches the reported CENPF band and its 357.5 kDa predicted mass |
| Band just below the precursor position | could reflect removal of the three-residue C-terminal propeptide; identity needs confirmation |
| Closely spaced bands near 358 kDa | could reflect different phosphorylation states; distinct migration is not established |
| Little or no band in a nucleolar fraction | fits the reported exclusion of CENPF from the nucleolus |
💡Expected CENPF appearanceCENPF has a predicted mass of 357.5 kDa and an observed whole-cell band at approximately 358 kDa; confirm band identity with appropriate antibody and sample controls.
| UniProt predicted mass | places the full-length protein near 357.5 kDa; the reported band is approximately 358 kDa |
| 3114-residue sequence | contributes to the large predicted precursor mass |
| C-terminal propeptide at residues 3112–3114 | cleavage would make the mature protein only slightly smaller than the precursor |
| Documented phosphorylation sites | could affect apparent mobility, but a measurable shift is not established |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | inefficient detection or transfer of this large protein | check transfer near 358 kDa and compare with a positive whole-cell lysate |
| Band higher than expected | identity or migration of the high-mass band is uncertain | check the high-mass ladder and confirm with an independent CENPF antibody |
| Band lower than expected | the three-residue propeptide cannot explain a large size difference | check for degradation and confirm the band with an independent CENPF antibody |
| Multiple bands | phosphorylation states are possible, but distinct band migration is unproven | compare phosphatase-treated and untreated samples and confirm band identity |
| Weak or no signal | a 358 kDa protein may transfer inefficiently | inspect transfer at high molecular weights and use a positive lysate |
| Fragments below expected size | sample degradation is possible; fragment masses are not supplied | prepare fresh lysate with protease inhibitors and compare antibody epitopes |
Experiment evidence
Western blot images showing CENPF band
A03311-1 · CENPF
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for CENPF Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for CENPF in Western blot, you can use appendix tissue lysate.
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 alongside the samples.
⚠️Feasibility: CENPF relocalizes during mitosis, so its signal may vary with the sample’s mitotic fraction.
HPA tissue expression evidence for CENPF
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 | germinal center cells | Medium | Protein (IHC) | HPA → |
| Colon | enterocytes | Medium | Protein (IHC) | HPA → |
| Duodenum | enterocytes | Medium | Protein (IHC) | HPA → |
| Endometrium | glandular cells | Medium | Protein (IHC) | HPA → |
| Epididymis | 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 → |
| Adrenal gland | glandular cells | Not detected | Protein (IHC) | HPA → |
| Breast | adipocytes | Not detected | Protein (IHC) | HPA → |
| Bronchus | respiratory epithelial cells | Not detected | Protein (IHC) | HPA → |
| Caudate | glial cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced CENPF Western Blot Tips
Deeper troubleshooting and optimisation questions for CENPF, answered from its protein features.
How should CENPF 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 CENPF isoforms explain multiple bands?
Isoforms · Only one isoform is supplied, with no alternative sequence. Do not assign additional bands to isoforms from these features alone; compare them with the expected approximately 358 kDa band.
Which CENPF modifications matter when interpreting bands?
PTM · UniProt lists 39 modified residues, including phosphoserine 2900, phosphothreonine 2949, N6-acetyllysine 2779, and a cysteine methyl ester at 3111. These are UniProt sequence coordinates; antibody or paper numbering may differ. Their presence alone does not establish a visible shift.
Does this guide establish induction of CENPF?
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 CENPF Western blot?
Transfer · CENPF is a large, 3114-residue protein predicted at 357.5 kDa. Check transfer at the high-mass end with an appropriate molecular-weight marker, and optimize transfer conditions if the approximately 358 kDa region transfers poorly.
How should blocking be optimized?
Blocking · Standard workflow guidance: follow the A03311-1 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 CENPF bands be quantified across samples?
Quantitation · Quantify the band near 358 kDa using comparable sample preparation and cell-cycle states. CENPF occupies nuclear, perinuclear, kinetochore, and spindle locations, so differences in fractionation or mitotic composition could affect comparisons.
Should CENPF migrate above its predicted mass?
Interpretation · CENPF is predicted at 357.5 kDa, and the supplied observed band is about 358 kDa. These values agree closely. Its listed modifications do not, by themselves, establish a visible band shift.
CENPF is linked to the cell cycle and mitosis. It relocalizes to kinetochores, centromeres, and the spindle during mitosis, whereas it is observed in the nucleus during interphase. Record cell-cycle state when comparing samples; these features do not specify an induction treatment.
The supplied features identify one isoform and a propeptide at UniProt residues 3112–3114, but do not establish that a lower band is a processed product. Check whether the expected approximately 358 kDa band is present and whether transfer or sample preparation differs before assigning an identity.
CENPF is linked to the cell cycle and mitosis. It relocalizes to kinetochores, centromeres, and the spindle during mitosis, whereas it is observed in the nucleus during interphase. Record cell-cycle state when comparing samples; these features do not specify an induction treatment.
The supplied features identify one isoform and a propeptide at UniProt residues 3112–3114, but do not establish that a lower band is a processed product. Check whether the expected approximately 358 kDa band is present and whether transfer or sample preparation differs before assigning an identity.
Boster reagents
CENPF 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-CENPF Antibody Picoband®
Cat # A03311-1
The catalog reports one anti-CENPF antibody, A03311-1, with a Western blot image from human HeLa whole-cell lysate. The caption reports a band near the expected 358 kDa using 0.5 μg/mL primary antibody. No publication or independent validation evidence is supplied.
Which to pick: A03311-1 is the only listed option. Its stated reactivity is human, and its WB image uses human HeLa lysate; the caption reports a 358 kDa band. Evidence for other samples or species is not supplied.
Source: BosterBio CENPF gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry P49454.
- Human Protein Atlas. CENPF tissue expression.
- PMC11344725 — target-verified WB comparison
- PMC8525945 — target-verified WB comparison
- PMC11807140 — target-verified WB comparison