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- Table of Contents
PFKP / ATP-dependent 6-phosphofructokinase, platelet type · Western blot design guide
Design a Western Blot for PFKP
Real validated PFKP Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-PFKP 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
PFKP 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 | ~85.6 kDa | |
| Gel | 8–10% (standard starting point) | |
| Positive control | Adrenal gland (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 + Phosphorylated | |
| Caveat | Phosphorylation-state controls | |
| Gene-set association | MSigDB Hallmark membership | |
| Isoform | 2 isoform(s) |
Section 1
Real Curated PFKP Western Blot Protocols
The A30471 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | HuvEc cells (catalog A30471) |
| Gel % | 8–10% (standard starting point) |
| Load | 20–30 µg total protein per lane; optimize for abundance (standard starting point) |
| Transfer | Standard semi-dry transfer; verify efficiency (standard starting point) |
| Membrane | 0.45 µm PVDF (standard starting point) |
| Blocking | 5% milk or 5% BSA in TBST (standard starting point) |
| Primary antibody | A30471; use the WB datasheet starting dilution (standard starting point) |
| Primary incubation | Overnight at 4 °C (standard starting point) |
| Secondary antibody | Species-matched HRP conjugate at validated dilution (standard starting point) |
| Secondary incubation | 1 h at room temperature (standard starting point) |
| Wash | 3 × 5 min in TBST (standard starting point) |
| Detection | ECL; bracket exposures to avoid saturation (standard starting point) |
Section 2
What Is the Expected PFKP Western Blot Band Size?
PFKP is predicted at 85.6 kDa; isoforms and O-GlcNAc are possible influences, but no empirical migration or distinct band pattern is supplied.
| Band near 85.6 kDa | Consistent with the predicted PFKP mass; identity requires confirmation |
| Single band near 85.6 kDa | Isoforms 1 and 2 may not resolve as separate bands |
| Multiple bands | Could reflect isoforms 1 and 2, but their migration difference is unknown |
| Slight mobility variation | Modification at O-GlcNAc serine 540 is possible; a visible shift is unproven |
💡Expected PFKP appearancePFKP has a predicted mass of 85.6 kDa, but no empirical band size is supplied; use antibody specificity and appropriate band-identity controls to assess a band near that size.
| UniProt predicted mass | Places the unmodified full-length protein near 85.6 kDa |
| O-GlcNAc at serine 540 | May affect migration, but a visible shift is not established |
| Isoform 1 | Its size relative to isoform 2 is not supplied |
| Isoform 2 | Its size relative to isoform 1 is not supplied |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | PFKP is cytoplasmic, so absent signal may reflect low abundance or failed detection | Check lysate loading and a positive control |
| Band higher than expected | A migration effect from O-GlcNAc at serine 540 is possible but unproven | Confirm identity with antibody controls and an independent antibody |
| Band lower than expected | Isoform size differences are unknown; a lower band is not assigned by these features | Check identity with an independent antibody |
| Multiple bands | Isoforms 1 and 2 could contribute, but separate migration is unproven | Use isoform-specific evidence or independent antibody controls |
| Weak or no signal | PFKP abundance or antibody detection may be limiting | Check loading, antibody performance, and a positive control |
Experiment evidence
Western blot images showing PFKP band
A30471 · PFKP
A30471 · PFKP
A30471 · PFKP
A07337-2 · PFKP
A07337-3 · PFKP
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for PFKP Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for PFKP in Western blot, you can use adrenal gland tissue, which HPA scores High.
Positive control: Adrenal gland (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: Cytoplasmic PFKP has a high-expression tissue and an HPA not-detected tissue available as controls.
HPA tissue expression evidence for PFKP
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 |
|---|---|---|---|---|
| Adrenal gland | glandular cells | High | Protein (IHC) | HPA → |
| Appendix | glandular cells | High | Protein (IHC) | HPA → |
| Breast | glandular cells | High | Protein (IHC) | HPA → |
| Bronchus | respiratory epithelial cells | High | Protein (IHC) | HPA → |
| Caudate | glial cells | High | 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 → |
| Liver | hepatocytes | Low | Protein (IHC) | HPA → |
| Bone marrow | hematopoietic cells | Medium | Protein (IHC) | HPA → |
| Cervix | glandular cells | Medium | Protein (IHC) | HPA → |
| Duodenum | glandular cells | Medium | Protein (IHC) | HPA → |
Section 3
Advanced PFKP Western Blot Tips
Deeper troubleshooting and optimisation questions for PFKP, answered from its protein features.
How should PFKP 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 PFKP isoforms produce different bands?
Isoforms · UniProt lists isoforms 1 and 2. Isoform 2 replaces canonical residues 1–87 with a different N-terminal sequence. Check whether the antibody recognizes both isoforms before assigning bands to them; the supplied features do not establish their apparent migration.
Which PFKP phosphorylation sites matter when interpreting a band?
PTM · The supplied canonical UniProt coordinates list phosphoserine at 6, 12, 21, 142, 386 and 783, and phosphotyrosine at 651. Sites 6, 12 and 21 fall within the segment replaced in isoform 2, so do not silently apply those coordinates to its altered sequence. Site presence does not prove a visible band shift.
Canonical UniProt annotations list N-acetylmethionine at position 1 and N6-acetyllysine at 395, 486 and 688. Position 1 lies in the sequence replaced in isoform 2. Record which isoform and numbering convention an antibody or paper uses before comparing sites; these annotations alone do not predict a separate band.
Yes. UniProt lists O-linked GlcNAc on serine 540 in canonical coordinates. Keep that site in mind when interpreting modification-sensitive results, but do not assign an unexpected band or mass difference to glycosylation without further evidence.
Canonical UniProt annotations list N-acetylmethionine at position 1 and N6-acetyllysine at 395, 486 and 688. Position 1 lies in the sequence replaced in isoform 2. Record which isoform and numbering convention an antibody or paper uses before comparing sites; these annotations alone do not predict a separate band.
Yes. UniProt lists O-linked GlcNAc on serine 540 in canonical coordinates. Keep that site in mind when interpreting modification-sensitive results, but do not assign an unexpected band or mass difference to glycosylation without further evidence.
Does this guide establish induction of PFKP?
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 PFKP?
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 A30471 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 PFKP be quantified?
Quantitation · Standard workflow guidance: quantify only a validated target band within the linear exposure range. Use consistent sample preparation and loading, retain biological replicates, and avoid interpreting saturation or loading differences as regulation.
Where should PFKP migrate on a Western blot?
Interpretation · Use 85.6 kDa as the predicted reference for PFKP. No observed band size is supplied, so check the marker position and report the measured apparent mass. The listed modifications alone do not establish a visible shift or explain a difference from 85.6 kDa.
Band intensity can be used to compare detected PFKP signal across samples if the antibody recognizes the forms being compared. PFKP functions as a tetramer and is annotated as an allosteric enzyme; band intensity alone does not measure its enzymatic activity or tetramer composition.
Check each band's apparent mass against the 85.6 kDa prediction and consider the annotated N-terminal sequence difference between isoforms 1 and 2. PFKP also has listed phosphorylation, acetylation and O-GlcNAc sites. None of those features alone identifies an extra band; verify antibody recognition and report bands without assigning an unsupported cause.
Band intensity can be used to compare detected PFKP signal across samples if the antibody recognizes the forms being compared. PFKP functions as a tetramer and is annotated as an allosteric enzyme; band intensity alone does not measure its enzymatic activity or tetramer composition.
Check each band's apparent mass against the 85.6 kDa prediction and consider the annotated N-terminal sequence difference between isoforms 1 and 2. PFKP also has listed phosphorylation, acetylation and O-GlcNAc sites. None of those features alone identifies an extra band; verify antibody recognition and report bands without assigning an unsupported cause.
Boster reagents
PFKP 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-K6PP Antibody
Cat # A30471
Real WB data
Anti-PFKP Antibody
Cat # A07337-2
Real WB data
Anti-PFKP Antibody Picoband®
Cat # A07337-3
All three listed anti-PFKP antibodies have WB images. A07337-2 shows an approximately 86 kDa band in human, rat, and mouse cell lysates; A07337-3 shows one in human lysates. A30471 shows a HuvEc cell blot. No independent validation is supplied.
Which to pick: Choose A07337-2 for the most directly illustrated sample range: human, rat, and mouse lysates. A07337-3 has illustrated human lysates; A30471 has a HuvEc blot and lists monkey reactivity, but its caption does not show a monkey sample.
Source: BosterBio PFKP gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry Q01813.
- Human Protein Atlas. PFKP tissue expression.
- PMC9972587 — target-verified WB comparison
- PMC10713276 — target-verified WB comparison
- PMC11628322 — target-verified WB comparison