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- Table of Contents
PFKM / ATP-dependent 6-phosphofructokinase, muscle type · Western blot design guide
Design a Western Blot for PFKM
Real validated PFKM Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-PFKM 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
PFKM 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 | ~85.2 kDa | |
| Observed band | ~85 kDa | |
| Gel | 5–20% (catalog A00437-2) | |
| 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 | Modification-state controls | |
| Gene-set association | MSigDB Hallmark membership | |
| Isoform | 3 isoform(s) |
Section 1
Real Curated PFKM Western Blot Protocols
The A00437-2 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | human Hela, human HepG2, human 293T (catalog A00437-2) |
| Gel % | 5–20% (catalog A00437-2) |
| Load | 30 ug; reducing conditions (catalog A00437-2) |
| Transfer | a nitrocellulose membrane at 150 mA for 50-90 minutes (catalog A00437-2) |
| Membrane | nitrocellulose membrane (catalog A00437-2) |
| Blocking | 5% non-fat milk/TBS for 1.5 hour at RT (catalog A00437-2) |
| Primary antibody | A00437-2 · 0.5 μg/mL (catalog A00437-2) |
| Primary incubation | overnight at 4°C (catalog A00437-2) |
| Secondary antibody | goat anti-rabbit IgG-HRP, 1:5000 (catalog A00437-2) |
| Secondary incubation | 1.5 hour at RT (catalog A00437-2) |
| Wash | TBS-0.1%Tween 3 times with 5 minutes each (catalog A00437-2) |
| Detection | ECL (catalog A00437-2) |
Section 2
What Is the Expected PFKM Western Blot Band Size?
PFKM is predicted at 85.2 kDa and observed at ~85 kDa; the cause of the small difference is not established.
| Band at ~85 kDa | Matches the empirical PFKM band and is close to the 85.2 kDa prediction. |
| Single sharp band near 85 kDa | Consistent with PFKM without a resolved modification or isoform shift. |
| Several bands near the expected region | Could reflect isoforms 1, 2, and 3; their migration differences are unestablished. |
| No separate glycosylated band | O-linked GlcNAc at Ser530 does not necessarily produce a visible shift. |
💡Expected PFKM appearancePFKM is predicted at 85.2 kDa and observed at ~85 kDa; confirm band identity with knockdown or an independent antibody because its listed features do not establish a distinct migration shift.
| Predicted 85.2 kDa mass | Corresponds closely to the observed ~85 kDa band. |
| O-linked GlcNAc at Ser530 | May affect mobility, but a visible shift is not established. |
| Splice isoform 1 | Its relative size and migration versus the other isoforms are unspecified. |
| Splice isoform 2 | Its relative size and migration versus the other isoforms are unspecified. |
| Splice isoform 3 | Its relative size and migration versus the other isoforms are unspecified. |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | Cytoplasmic PFKM may be below detection in the tested lysate. | Check loading and antibody performance with a positive lysate. |
| Band higher than expected | The listed features do not establish a higher-migrating PFKM band. | Check specificity with knockdown or an independent antibody. |
| Band lower than expected | An isoform is possible, but its migration is unspecified. | Confirm identity with knockdown or an independent antibody. |
| Multiple bands | Isoforms 1, 2, and 3 are listed, but distinct bands are unverified. | Compare bands after PFKM knockdown. |
| Weak or no signal | PFKM signal may be below detection in the tested sample. | Check protein loading and include a positive lysate. |
| Fragments below expected size | Sample degradation may produce smaller reactive fragments. | Prepare fresh lysate with protease inhibitors and confirm band identity. |
Experiment evidence
Western blot images showing PFKM band
A00437-2 · PFKM
M04370-1 · PFKM
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for PFKM Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for PFKM in Western blot, you can use adrenal gland tissue lysate.
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 alongside the samples.
⚠️Feasibility: Cytoplasmic PFKM should be accessible in tissue lysates, with adipose tissue providing an HPA not-detected control.
HPA tissue expression evidence for PFKM
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 → |
| Bone marrow | hematopoietic cells | High | Protein (IHC) | HPA → |
| Bronchus | respiratory epithelial cells | High | Protein (IHC) | HPA → |
| Caudate | neuronal 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 → |
| Esophagus | squamous epithelial cells | Not detected | Protein (IHC) | HPA → |
| Oral mucosa | squamous epithelial cells | Low | Protein (IHC) | HPA → |
| Breast | glandular cells | Medium | Protein (IHC) | HPA → |
| Cervix | squamous epithelial cells | Medium | Protein (IHC) | HPA → |
Section 3
Advanced PFKM Western Blot Tips
Deeper troubleshooting and optimisation questions for PFKM, answered from its protein features.
How should PFKM 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 PFKM isoforms produce additional bands?
Isoforms · UniProt lists three isoforms. Isoform 2 lacks residues 282–312, while isoform 3 replaces the canonical N-terminal methionine with a longer sequence. Either sequence change could affect migration; band position alone cannot identify an isoform.
Which PFKM modifications should I consider when interpreting bands?
PTM · In canonical UniProt numbering, PFKM has N-acetylthreonine 2; phosphoserines 133, 377, 667, and 775; and N6-(2-hydroxyisobutyryl)lysine 557. These sites identify possible modification-dependent forms but do not establish that separate bands will be visible.
UniProt lists one O-linked GlcNAc site, serine 530 in canonical numbering. Its presence alone does not establish a detectable band shift. Do not assign a shifted band to this modification from migration alone.
UniProt lists one O-linked GlcNAc site, serine 530 in canonical numbering. Its presence alone does not establish a detectable band shift. Do not assign a shifted band to this modification from migration alone.
Does this guide establish induction of PFKM?
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 PFKM?
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 A00437-2 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.
What does a PFKM Western blot quantify?
Quantitation · A PFKM band can be used to compare detected protein abundance across matched samples. PFKM is cytoplasmic and functions in tetramers containing different phosphofructokinase subunits; band intensity alone does not measure tetramer composition or enzyme activity.
Does the observed PFKM band match its predicted mass?
Interpretation · The observed band near 85 kDa agrees with the predicted 85.2 kDa. The listed modifications alone do not establish a visible shift or explain any small difference in apparent mass.
Consider the documented isoform 2 deletion, isoform 3 N-terminal replacement, and listed modification sites when reviewing band positions. The canonical protein is predicted at 85.2 kDa, but these features alone cannot identify an unexpected band. Confirm its identity with an independent assay.
Consider the documented isoform 2 deletion, isoform 3 N-terminal replacement, and listed modification sites when reviewing band positions. The canonical protein is predicted at 85.2 kDa, but these features alone cannot identify an unexpected band. Confirm its identity with an independent assay.
Boster reagents
PFKM 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-PFKM Antibody Picoband®
Cat # A00437-2
Real WB data
Anti-Fructose 6 Phosphate Kinase Rabbit Monoclonal Antibody
Cat # M04370-1
Both listed anti-PFKM antibodies have Western blot images using human cell lysates and rat and mouse tissues. A00437-2 shows an approximately 85 kDa band; M04370-1 shows approximately 81 kDa, compared with the stated 85 kDa expectation.
Which to pick: Match the pictured samples to your experiment: A00437-2 includes HeLa, HepG2, 293T, U-87MG, and rodent heart; M04370-1 includes HeLa, MCF-7, SH-SY5Y, and rodent heart and brain. Both list human, mouse, and rat reactivity.
Source: BosterBio PFKM gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry P08237.
- Human Protein Atlas. PFKM tissue expression.
- PMC12288408 — target-verified WB comparison
- PMC11670517 — target-verified WB comparison
- PMC8058737 — target-verified WB comparison