This website uses cookies to ensure you get the best experience on our website.
- Table of Contents
PFKFB4 / 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 4 · Western blot design guide
Design a Western Blot for PFKFB4
Source-linked PFKFB4 Western blot protocol options, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-PFKFB4 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
PFKFB4 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 | ~54 kDa | |
| Observed band | ~54 kDa | |
| Gel | 5–20% (catalog A07028-1) | |
| Negative control | Suggested KO / knockdown lysate |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Phosphorylated | |
| Caveat | Phosphorylation-state controls | |
| Gene-set association | MSigDB C7 membership | |
| Isoform | 3 isoform(s) |
Section 1
Source-Linked PFKFB4 Western Blot Protocol Options
The A07028-1 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | human 293T, human Raji, human PC-3 (catalog A07028-1) |
| Gel % | 5–20% (catalog A07028-1) |
| Load | 30 ug; reducing conditions (catalog A07028-1) |
| Transfer | a nitrocellulose membrane at 150 mA for 50-90 minutes (catalog A07028-1) |
| Membrane | nitrocellulose membrane (catalog A07028-1) |
| Blocking | 5% non-fat milk/TBS for 1.5 hour at RT (catalog A07028-1) |
| Primary antibody | A07028-1 · 0.5 μg/mL (catalog A07028-1) |
| Primary incubation | overnight at 4°C (catalog A07028-1) |
| Secondary antibody | goat anti-rabbit IgG-HRP, 1:5000 (catalog A07028-1) |
| Secondary incubation | 1.5 hour at RT (catalog A07028-1) |
| Wash | TBS-0.1%Tween 3 times with 5 minutes each (catalog A07028-1) |
| Detection | ECL (catalog A07028-1) |
Section 2
What Is the Expected PFKFB4 Western Blot Band Size?
PFKFB4 is predicted at 54 kDa and observed at ~54 kDa; homodimerization and isoforms may affect other bands, but their migration is unestablished.
| Band at ~54 kDa | Matches the empirical PFKFB4 band and predicted mass; confirm identity with antibody controls |
| Band near ~108 kDa | Could reflect retained PFKFB4 homodimer; its migration at this size is unconfirmed |
| Several bands at different positions | Could reflect isoforms 1, 2, and 3; their individual sizes are unknown |
| One band despite three isoforms | The isoforms need not produce resolvable bands |
💡Expected PFKFB4 appearancePFKFB4 is predicted at 54 kDa and observed at ~54 kDa in reducing whole-cell lysates; confirm band identity with ordinary antibody and sample controls.
| UniProt predicted mass | 54 kDa predicted; the empirical band is ~54 kDa |
| Homodimer formation | Could yield a band near twice the monomer size if the dimer remains intact during analysis |
| Isoform 1 | Individual apparent size is not supplied |
| Isoforms 2 and 3 | May differ in size from isoform 1, but individual masses and resolvable shifts are not supplied |
| Situation | Likely cause | Next action |
|---|---|---|
| Band higher than expected | Possible retained homodimer or unidentified band | Compare reducing and nonreducing samples and verify band identity |
| Band lower than expected | Possible alternative isoform or sample degradation; identity unconfirmed | Check isoform coverage and repeat with fresh, protected lysate |
| Multiple bands | Possible isoforms 1, 2, and 3; their migration is unknown | Check antibody epitope coverage and validate bands with PFKFB4 depletion |
| Weak or no signal | Low PFKFB4 abundance or insufficient antibody sensitivity | Check loading and use a validated positive-control lysate |
| Fragments below expected size | Possible sample degradation | Prepare fresh lysate with protease inhibitors and verify band identity |
Experiment evidence
Western blot images showing PFKFB4 band
A07028-1 · PFKFB4
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for PFKFB4 Western blot
🧪For positive controls for PFKFB4 in Western blot, you can use a sample with independently verified PFKFB4 expression; the supplied HPA evidence identifies no positive tissue or cell line.
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 alongside the samples.
⚠️Feasibility: HPA provides no tissue data, so tissue controls cannot be selected from this evidence.
HPA tissue expression evidence for PFKFB4
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 PFKFB4 Western Blot Tips
Deeper troubleshooting and optimisation questions for PFKFB4, answered from its protein features.
How should PFKFB4 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 PFKFB4 isoforms produce lower bands?
Isoforms · Possibly. Isoform 2 lacks canonical residues 330–364, and isoform 3 lacks residues 1–34. These deletions could change migration, but the features do not establish where either isoform runs on a blot.
Check the antibody epitope against the deleted regions. An epitope within canonical residues 330–364 would be absent from isoform 2; one within residues 1–34 would be absent from isoform 3.
Use an antibody whose epitope is retained in all three isoforms, then quantify any validated PFKFB4 bands consistently. An antibody targeting residues 1–34 or 330–364 could miss isoform 3 or isoform 2, respectively.
Check the antibody epitope against the deleted regions. An epitope within canonical residues 330–364 would be absent from isoform 2; one within residues 1–34 would be absent from isoform 3.
Use an antibody whose epitope is retained in all three isoforms, then quantify any validated PFKFB4 bands consistently. An antibody targeting residues 1–34 or 330–364 could miss isoform 3 or isoform 2, respectively.
Which PFKFB4 phosphorylation site is listed?
PTM · UniProt lists phosphothreonine at canonical residue 444, modified by PKC. Check the numbering convention when comparing antibody or paper descriptions, since their residue numbers may differ.
Thr444 phosphorylation by PKC is listed, but that feature alone does not demonstrate a visible band shift or explain a difference between apparent and predicted mass. Test phosphorylation directly before assigning a shifted band to this modification.
Thr444 phosphorylation by PKC is listed, but that feature alone does not demonstrate a visible band shift or explain a difference between apparent and predicted mass. Test phosphorylation directly before assigning a shifted band to this modification.
Does this guide establish induction of PFKFB4?
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 PFKFB4?
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 A07028-1 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 PFKFB4 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.
Is a band near 54 kDa expected for PFKFB4?
Interpretation · Yes. The supplied predicted mass and observed Western blot band are both approximately 54 kDa. The listed features alone do not establish a visible shift from that mass.
PFKFB4 is annotated as a homodimer, so a higher band may warrant investigation. The annotation does not establish that dimers persist during Western blot preparation or identify any observed higher band.
PFKFB4 is annotated as a homodimer, so a higher band may warrant investigation. The annotation does not establish that dimers persist during Western blot preparation or identify any observed higher band.
Boster reagents
PFKFB4 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-PFKFB4 Antibody Picoband®
Cat # A07028-1
The listed anti-PFKFB4 antibody, A07028-1, is reported reactive with human samples. Its Western blot image shows an approximately 54 kDa band in human 293T, Raji, and PC-3 whole cell lysates. The supplied evidence covers these tested samples only.
Which to pick: A07028-1 is the only listed option. Choose it for human PFKFB4 Western blots when the documented 293T, Raji, and PC-3 lysate results are relevant to your experiment.
Source: BosterBio PFKFB4 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry Q16877.
- Human Protein Atlas. PFKFB4 tissue expression.
- PMC8176253 — target-verified WB comparison
- PMC8692682 — target-verified WB comparison
- PMC10140800 — target-verified WB comparison