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PFKFB3 / 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 · Western blot design guide
Design a Western Blot for PFKFB3
Real validated PFKFB3 Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-PFKFB3 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
PFKFB3 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 | ~59.6 kDa | |
| Observed band | ~58 kDa | |
| Gel | 10% (catalog M02382) | |
| Positive control | Lung (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 | |
| Caveat | Phosphorylation-state controls | |
| Gene-set association | MSigDB Hallmark membership | |
| Isoform | 4 isoform(s) |
Section 1
Real Curated PFKFB3 Western Blot Protocols
The M02382 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | human Hela, human MCF-7, human A549 (catalog M02382) |
| Gel % | 10% (catalog M02382) |
| Load | 30 ug; reducing conditions (catalog M02382) |
| Transfer | a nitrocellulose membrane at 150 mA for 50-90 minutes (catalog M02382) |
| Membrane | nitrocellulose membrane (catalog M02382) |
| Blocking | 5% non-fat milk/TBS for 1.5 hour at RT (catalog M02382) |
| Primary antibody | M02382 · 1:500 (catalog M02382) |
| Primary incubation | overnight at 4°C (catalog M02382) |
| Secondary antibody | goat anti-rabbit IgG-HRP, 1:5000 (catalog M02382) |
| Secondary incubation | 1.5 hour at RT (catalog M02382) |
| Wash | TBS-0.1%Tween 3 times with 5 minutes each (catalog M02382) |
| Detection | ECL (catalog M02382) |
Section 2
What Is the Expected PFKFB3 Western Blot Band Size?
PFKFB3 is predicted at 59.6 kDa and observed near 58 kDa in reducing whole-cell lysates; the cause of the difference is not established.
| Band near 58 kDa | Empirical PFKFB3 band in reducing whole-cell lysates; confirm identity with appropriate controls |
| Band near 59.6 kDa | Near the predicted mass of a PFKFB3 monomer |
| Higher band near twice the monomer size | Could reflect a homodimer if it survives sample preparation |
| Several bands at different positions | Could reflect isoforms 1, 2, 3, and 4; distinct migration is unverified |
💡Expected PFKFB3 appearancePFKFB3 has a predicted mass of 59.6 kDa and an empirical band near 58 kDa in reducing whole-cell lysates; confirm band identity with a positive control or PFKFB3 depletion.
| Predicted PFKFB3 mass | The 59.6 kDa sequence mass is close to the empirical band near 58 kDa |
| PFKFB3 homodimer | Could appear near twice the monomer size if the dimer survives sample preparation; SDS stability is unestablished |
| Isoform 1 | May migrate differently from other isoforms; its individual mass is unavailable |
| Isoform 2 | May migrate differently from other isoforms; its individual mass is unavailable |
| Isoforms 3 and 4 | May differ in migration, but their individual masses and separation are unestablished |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | PFKFB3 may be below detection in the tested sample | Compare with a lysate that yields the reported 58 kDa band and check loading and transfer |
| Band higher than expected | A PFKFB3 homodimer may persist during sample preparation | Compare fully denatured reducing samples and verify the band by PFKFB3 depletion |
| Band lower than expected | The reported 58 kDa band runs slightly below the 59.6 kDa predicted mass; the cause is unestablished | Compare with the reported band and verify identity by PFKFB3 depletion |
| Multiple bands | Isoforms 1, 2, 3, and 4 are documented, but their separation on a blot is unestablished | Check which bands decrease after PFKFB3 depletion |
| Weak or no signal | PFKFB3 abundance or assay sensitivity may be insufficient | Use a positive-control lysate and check antibody conditions and transfer |
Experiment evidence
Western blot images showing PFKFB3 band
M02382 · PFKFB3
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for PFKFB3 Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for PFKFB3 in Western blot, you can use lung tissue, which HPA rates High.
Positive control: Lung (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: A tissue negative control is feasible because HPA reports PFKFB3 as not detected in adipose tissue.
HPA tissue expression evidence for PFKFB3
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 |
|---|---|---|---|---|
| Lung | macrophages | High | Protein (IHC) | HPA → |
| Placenta | trophoblastic cells | High | Protein (IHC) | HPA → |
| Stomach | glandular cells | High | Protein (IHC) | HPA → |
| Adrenal gland | glandular cells | Medium | Protein (IHC) | HPA → |
| Appendix | 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 → |
| Ovary | ovarian stroma cells | Not detected | Protein (IHC) | HPA → |
| Skeletal muscle | myocytes | Not detected | Protein (IHC) | HPA → |
| Cerebral cortex | glial cells | Low | Protein (IHC) | HPA → |
| Gallbladder | glandular cells | Low | Protein (IHC) | HPA → |
Section 3
Advanced PFKFB3 Western Blot Tips
Deeper troubleshooting and optimisation questions for PFKFB3, answered from its protein features.
How should PFKFB3 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 PFKFB3 isoforms produce different bands?
Isoforms · UniProt lists four isoforms. Isoforms 3 and 4 replace canonical residues 1–26 with different N-terminal sequences; isoform 2 replaces residues 506–520. These sequence differences may affect migration, but they do not establish which isoform produced a band.
Check the antibody epitope against the altered N terminus in isoforms 3 and 4 and the altered C terminus in isoform 2. An antibody targeting either region may recognize isoforms differently.
Check the antibody epitope against the altered N terminus in isoforms 3 and 4 and the altered C terminus in isoform 2. An antibody targeting either region may recognize isoforms differently.
Which PFKFB3 phosphorylation sites matter when interpreting bands?
PTM · UniProt lists phosphoserine 461 by AMPK, phosphothreonine 463, phosphoserine 467, and phosphothreonine 471 by PKC. These are canonical UniProt coordinates; antibody or paper numbering may differ. Their presence alone does not establish a visible band shift.
For a phosphosite-specific signal, compare it with total PFKFB3 measured in the same samples. Confirm that the phosphosite antibody uses the intended UniProt coordinate and that the total antibody recognizes the isoforms being compared. The feature list does not establish phosphorylation occupancy.
For a phosphosite-specific signal, compare it with total PFKFB3 measured in the same samples. Confirm that the phosphosite antibody uses the intended UniProt coordinate and that the total antibody recognizes the isoforms being compared. The feature list does not establish phosphorylation occupancy.
Does this guide establish induction of PFKFB3?
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 PFKFB3?
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 M02382 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 PFKFB3 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.
How should the ~58 kDa band compare with predicted PFKFB3 mass?
Interpretation · The observed ~58 kDa band is close to the 59.6 kDa predicted mass. Apparent migration can differ from calculated mass; the listed features do not establish why this band runs at ~58 kDa.
PFKFB3 forms a homodimer and can form a heterodimer with PFKFB2. These interactions make complexes a possibility to investigate, but they do not identify a higher-mass Western blot band. Check band identity experimentally before assigning it to either complex.
PFKFB3 forms a homodimer and can form a heterodimer with PFKFB2. These interactions make complexes a possibility to investigate, but they do not identify a higher-mass Western blot band. Check band identity experimentally before assigning it to either complex.
Boster reagents
PFKFB3 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-PFKFB3 Monoclonal Antibody
Cat # M02382
the supplier M02382 is an anti-PFKFB3 monoclonal antibody with a Western blot image showing a band near 58 kDa (expected 60 kDa) in human HeLa, MCF-7, A549, and U251 whole cell lysates. The supplied image does not show mouse or rat samples.
Which to pick: M02382 is the only listed antibody. Its reported reactivity includes human, mouse, and rat, but the supplied Western blot image uses human cell lines. For mouse or rat experiments, the supplied evidence offers no sample-specific Western blot example.
Source: BosterBio PFKFB3 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry Q16875.
- Human Protein Atlas. PFKFB3 tissue expression.
- PMC9474594 — target-verified WB comparison
- PMC10692378 — target-verified WB comparison
- PMC6921228 — target-verified WB comparison