This website uses cookies to ensure you get the best experience on our website.
- Table of Contents
GCK / Hexokinase-4 · Western blot design guide
Design a Western Blot for GCK
Real validated GCK Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-GCK 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
GCK 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 | ~52.2 kDa | |
| Gel | 12–15% (standard starting point) | |
| Positive control | Appendix (IHC candidate; verify WB) +4 more | |
| Negative control | Vagina (IHC candidate; verify WB) |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | — | |
| Caveat | — | |
| Gene-set association | MSigDB Hallmark membership | |
| Isoform | 3 isoform(s) |
Section 1
Real Curated GCK Western Blot Protocols
The M00884-2 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | BxPC-3 cell lysate (catalog M00884-2) |
| Gel % | 12–15% (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 | M00884-2; 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 GCK Western Blot Band Size?
GCK is predicted at 52.2 kDa; its isoforms could affect migration, but no distinct bands or empirical migration have been demonstrated here.
| Band near 52.2 kDa | Consistent with the predicted mass of GCK; identity requires controls |
| A single sharp band near 52.2 kDa | Consistent with one detectable GCK form in that sample |
| Doublet near the expected region | Could reflect isoforms if their migration and identity are verified |
| Several bands at different positions | Could reflect isoforms 1, 2, and 3 if independently identified |
💡Expected GCK appearanceUniProt predicts GCK at 52.2 kDa; no empirical band size or isoform migration is supplied, so confirm a band near that size with ordinary identity controls.
| UniProt predicted mass | Places the reference full-length GCK band near 52.2 kDa |
| Isoform 1 | Its individual mass and migration are not supplied |
| Isoform 2 | Its size relative to isoform 1 is not established |
| Isoform 3 | Its size relative to the other isoforms is not established |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | GCK may be distributed among cytoplasm, nucleus, and mitochondria | Check enriched fractions with compartment markers and a positive control |
| Band higher than expected | The band identity or isoform migration is unverified | Compare with a GCK positive control and an independent antibody |
| Band lower than expected | The band identity or isoform migration is unverified | Check with an independent antibody and a GCK depletion control |
| Multiple bands | GCK has isoforms 1, 2, and 3, but distinct migration is unproven | Test band identity with isoform-aware controls or GCK depletion |
| Fragments below expected size | The smaller signals have no established identity from the supplied features | Check sample integrity and whether the signals disappear after GCK depletion |
Experiment evidence
Western blot images showing GCK band
M00884-2 · GCK
A00884-1 · GCK
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for GCK Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for GCK in Western blot, you can use appendix tissue, which HPA scores as High.
Positive control: Appendix (IHC candidate; verify WB)
Negative control: Vagina (IHC candidate; verify WB)
Loading controls: Run GAPDH, β-actin, and a total-protein stain alongside the samples.
⚠️Feasibility: GCK is intracellular, and HPA identifies vagina as a tissue where it is not detected.
HPA tissue expression evidence for GCK
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 | glandular cells | High | Protein (IHC) | HPA → |
| Bronchus | respiratory epithelial cells | High | Protein (IHC) | HPA → |
| Cerebral cortex | neuronal cells | High | Protein (IHC) | HPA → |
| Colon | glandular cells | High | Protein (IHC) | HPA → |
| Duodenum | glandular cells | High | Protein (IHC) | HPA → |
Lower expression tissues · IHC evidence, not confirmed WB-negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Vagina | squamous epithelial cells | Not detected | Protein (IHC) | HPA → |
| Adrenal gland | glandular cells | Low | Protein (IHC) | HPA → |
| Cervix | glandular cells | Low | Protein (IHC) | HPA → |
| Fallopian tube | glandular cells | Low | Protein (IHC) | HPA → |
| Heart muscle | cardiomyocytes | Low | Protein (IHC) | HPA → |
Section 3
Advanced GCK Western Blot Tips
Deeper troubleshooting and optimisation questions for GCK, answered from its protein features.
Where should the main GCK band appear?
Band shift · The predicted mass is 52.2 kDa. No observed band position is supplied, so use 52.2 kDa as a reference rather than an expected exact migration position.
Could GCK isoforms produce different bands?
Isoforms · Three isoforms are listed. Isoforms 2 and 3 have alternative sequences at UniProt positions 1–15. These changes could affect antibody recognition, but the supplied features do not establish distinct visible band positions.
Check whether the antibody epitope overlaps UniProt positions 1–15, where isoforms 2 and 3 differ from the canonical sequence. An epitope in that region may recognize the isoforms differently.
Check whether the antibody epitope overlaps UniProt positions 1–15, where isoforms 2 and 3 differ from the canonical sequence. An epitope in that region may recognize the isoforms differently.
Does a listed modification explain a shifted GCK band?
PTM · The supplied features list no modified residues or glycosylation sites. They therefore provide no specific modification to assign to a shifted band. Feature presence alone would not establish a visible shift.
Does this guide establish induction of GCK?
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 GCK?
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 M00884-2 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.
How should GCK be quantified across cell fractions?
Quantitation · GCK is listed in the cytoplasm and nucleus, with mitochondrial localization also annotated. When comparing fractions, quantify bands within each fraction and account for fraction loading before interpreting a difference as a change in total GCK.
Do unexpected high-mass bands indicate GCK oligomers?
Interpretation · GCK is annotated as a monomer. The supplied features do not identify a stable GCK oligomer that would explain a high-mass band. Check antibody specificity before assigning that band to GCK.
Boster reagents
GCK 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-Glucokinase Rabbit Monoclonal Antibody
Cat # M00884-2
Real WB data
Anti-Glucokinase/GCK Antibody Picoband®
Cat # A00884-1
Two the supplier anti-GCK antibodies have Western blot images: M00884-2 in BxPC-3 lysate and A00884-1 in human cell, rat liver and cell, and mouse cell lysates. The supplied evidence provides product images but no publication or independent validation data.
Which to pick: For human BxPC-3 lysate, consider M00884-2. For the listed human, rat, or mouse sample types, consider A00884-1, whose image reports a band near 52 kDa. Both have WB images; choose based on your sample.
Source: BosterBio GCK gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry P35557.
- Human Protein Atlas. GCK tissue expression.
- PMC3859026 — target-verified WB comparison
- PMC12335957 — target-verified WB comparison