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- Table of Contents
HK3 / Hexokinase-3 · Western blot design guide
Design a Western Blot for HK3
Source-linked HK3 Western blot protocol options, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-HK3 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
HK3 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 | ~99 kDa | |
| Observed band | ~99 kDa | |
| Gel | 8% (catalog A05145-3) | |
| Positive control | Bone marrow (IHC candidate; verify WB) +3 more | |
| Negative control | Adipose tissue (IHC candidate; verify WB) |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | — | |
| Caveat | — | |
| Gene-set association | MSigDB C7 membership | |
| Isoform | 1 isoform(s) |
Section 1
Source-Linked HK3 Western Blot Protocol Options
The A05145-3 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | human Raji, human Ramos, human SH-SY5Y (catalog A05145-3) |
| Gel % | 8% (catalog A05145-3) |
| Load | 30 ug; reducing conditions (catalog A05145-3) |
| Transfer | a nitrocellulose membrane at 150 mA for 50-90 minutes (catalog A05145-3) |
| Membrane | nitrocellulose membrane (catalog A05145-3) |
| Blocking | 5% non-fat milk/TBS for 1.5 hour at RT (catalog A05145-3) |
| Primary antibody | A05145-3 · 0.5 μg/mL (catalog A05145-3) |
| Primary incubation | overnight at 4°C (catalog A05145-3) |
| Secondary antibody | goat anti-rabbit IgG-HRP, 1:5000 (catalog A05145-3) |
| Secondary incubation | 1.5 hour at RT (catalog A05145-3) |
| Wash | TBS-0.1%Tween 3 times with 5 minutes each (catalog A05145-3) |
| Detection | ECL (catalog A05145-3) |
Section 2
What Is the Expected HK3 Western Blot Band Size?
HK3 is predicted at 99 kDa and observed at approximately 99 kDa; confirm the band with standard identity controls.
| Single band near 99 kDa | Consistent with the predicted HK3 mass and the reported blot |
| Faint band near 99 kDa | Possible HK3 signal requiring identity confirmation |
| No band near 99 kDa | HK3 was not detected in this sample |
| Additional bands away from 99 kDa | Their identities are not established by the supplied features |
💡Expected HK3 appearanceHK3 has a predicted mass of 99 kDa and an observed band at approximately 99 kDa in reducing whole-cell lysates; confirm band identity with appropriate controls.
| UniProt predicted mass | Places full-length HK3 near 99 kDa |
| 99,025 Da calculated mass | Corresponds to approximately 99 kDa |
| Full-length predicted mass | Agrees with the reported approximately 99 kDa band |
| Reference protein mass | Provides no evidence for a distinct shifted band |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | HK3 signal is undetectable; the supplied features do not establish why | Check sample loading and transfer against a positive lysate |
| Band higher than expected | The supplied features do not explain migration above 99 kDa | Confirm identity with an independent HK3 antibody or depletion control |
| Band lower than expected | The supplied features do not establish a smaller HK3 product | Check sample integrity and confirm identity with an independent antibody |
| Multiple bands | Additional band identities are not established | Compare with an HK3 depletion control |
| Weak or no signal | Insufficient detectable signal or incomplete transfer | Check a positive lysate and verify transfer near 99 kDa |
Experiment evidence
Western blot images showing HK3 band
A05145-3 · HK3
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for HK3 Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for HK3 in Western blot, you can use bone marrow lysate, which HPA rates as high for HK3.
Positive control: Bone marrow (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: Adipose tissue is a plausible negative control based on HPA, but verify the absence of a band in your lysate.
HPA tissue expression evidence for HK3
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 |
|---|---|---|---|---|
| Bone marrow | hematopoietic cells | High | Protein (IHC) | HPA → |
| Lung | macrophages | High | Protein (IHC) | HPA → |
| Spleen | cells in red pulp | High | Protein (IHC) | HPA → |
| Lymph node | non-germinal center 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 → |
| Adrenal gland | glandular cells | Not detected | Protein (IHC) | HPA → |
| Appendix | glandular cells | Not detected | Protein (IHC) | HPA → |
| Breast | adipocytes | Not detected | Protein (IHC) | HPA → |
| Bronchus | respiratory epithelial cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced HK3 Western Blot Tips
Deeper troubleshooting and optimisation questions for HK3, answered from its protein features.
Where should the HK3 band appear?
Band shift · HK3 has a predicted mass of 99 kDa, and the supplied observed band is approximately 99 kDa. Use that region as the starting point for identifying the band.
Does HK3 have a documented isoform that explains another band?
Isoforms · The supplied UniProt record lists one isoform and no alternative sequence. It provides no isoform-based explanation for an additional band.
Do listed modifications predict an HK3 band shift?
PTM · No modified residues or glycosylation sites are listed. The supplied features therefore identify no modification that would predict a shift; feature listings alone cannot establish why a band migrates differently.
Does this guide establish induction of HK3?
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.
What transfer method to use for HK3 Western blot?
Transfer · Use the approximately 99 kDa target region to check whether transfer was effective. The supplied features do not specify a transfer method or settings.
How should blocking be optimized?
Blocking · Standard workflow guidance: follow the A05145-3 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 HK3 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 HK3 band intensity be interpreted?
Interpretation · Quantify the approximately 99 kDa band consistently across samples. HK3 is annotated as an allosteric enzyme involved in glycolysis, so band intensity reflects detected protein signal, not enzyme activity by itself.
Compare them with the expected approximately 99 kDa band. The supplied record lists no alternative sequence, modified residues, or glycosylation sites to assign an additional band to a specific HK3 form.
Compare them with the expected approximately 99 kDa band. The supplied record lists no alternative sequence, modified residues, or glycosylation sites to assign an additional band to a specific HK3 form.
Boster reagents
HK3 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-HK3 Antibody Picoband®
Cat # A05145-3
The listed anti-HK3 antibody, A05145-3, has a Western blot image using human Raji, Ramos, and SH-SY5Y whole-cell lysates. The caption reports a band near the expected 99 kDa. Evidence for other specimens or species is not supplied.
Which to pick: A05145-3 is the only listed option. For human lysates, its WB example used 30 µg per lane under reducing conditions and 0.5 µg/mL antibody. The named cell lines are tested examples, not universal validation.
Source: BosterBio HK3 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry P52790.
- Human Protein Atlas. HK3 tissue expression.
- PMC13460742 — target-verified WB comparison
- PMC13214176 — target-verified WB comparison
- PMC12105894 — target-verified WB comparison