This website uses cookies to ensure you get the best experience on our website.
- Table of Contents
HCK / Tyrosine-protein kinase HCK · Western blot design guide
Design a Western Blot for HCK
Real validated HCK Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-HCK 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
HCK 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 | ~60 kDa | |
| Gel | 10% (catalog A01073-3) | |
| Positive control | Bone marrow (IHC candidate; verify WB) +2 more | |
| Negative control | Adipose tissue (IHC candidate; verify WB) |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Phosphorylated + Ubl conjugation | |
| Caveat | Modification-state controls | |
| Gene-set association | MSigDB C7 membership | |
| Isoform | 4 isoform(s) |
Section 1
Real Curated HCK Western Blot Protocols
The A01073-3 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | human THP-1, rat spleen (catalog A01073-3) |
| Gel % | 10% (catalog A01073-3) |
| Load | 30 ug; reducing conditions (catalog A01073-3) |
| Transfer | a nitrocellulose membrane at 150 mA for 50-90 minutes (catalog A01073-3) |
| Membrane | nitrocellulose membrane (catalog A01073-3) |
| Blocking | 5% non-fat milk/TBS for 1.5 hour at RT (catalog A01073-3) |
| Primary antibody | A01073-3 · 0.5 μg/mL (catalog A01073-3) |
| Primary incubation | overnight at 4°C (catalog A01073-3) |
| Secondary antibody | goat anti-rabbit IgG-HRP, 1:5000 (catalog A01073-3) |
| Secondary incubation | 1.5 hour at RT (catalog A01073-3) |
| Wash | TBS-0.1%Tween 3 times with 5 minutes each (catalog A01073-3) |
| Detection | ECL (catalog A01073-3) |
Section 2
What Is the Expected HCK Western Blot Band Size?
HCK is predicted at 59.6 kDa and observed at ~60 kDa; the small difference has no established cause.
| Band at ~60 kDa | Matches the empirical HCK band and its 59.6 kDa predicted mass |
| Additional bands at other positions | Could reflect isoforms 1, 2, 3, or 4; their migration is not established |
| Doublet near ~60 kDa | Could reflect annotated phosphorylation, but a visible shift is not established |
| Bands in cytosolic and membrane fractions | Consistent with the reported distribution of isoform 1 |
💡Expected HCK appearanceHCK is predicted at 59.6 kDa and observed at ~60 kDa; confirm band identity with a positive control and an antibody specificity check, since no individual isoform has an assigned band size.
| UniProt predicted mass | 59.6 kDa predicted; the empirical band is ~60 kDa |
| Splice isoform 1 | May differ in size from other isoforms; its migration is not established |
| Splice isoform 2 | May differ in size from other isoforms; its migration is not established |
| Splice isoform 3 | May differ in size from other isoforms; its migration is not established |
| Splice isoform 4 | May differ in size from other isoforms; its migration is not established |
| Situation | Likely cause | Next action |
|---|---|---|
| Weak or no signal | HCK distribution between cytosolic and membrane compartments may affect a fractionated sample | Check whole-cell lysate and compare fractions with a positive control |
| Band higher than expected | An apparent shift is not established by the annotated modifications | Compare with a positive control and check antibody specificity |
| Band lower than expected | An isoform could differ in size, but no isoform band size is supplied | Verify the band with an independent HCK antibody or isoform-specific control |
| Multiple bands | Four isoforms and phosphorylation sites are annotated, but distinct bands are unverified | Use isoform-specific controls and compare samples with and without phosphatase treatment |
| Fragments below expected size | Their identity is not established by the supplied features | Check sample integrity and confirm HCK identity with an independent antibody |
Experiment evidence
Western blot images showing HCK band
A01073-3 · HCK
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for HCK Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for HCK in Western blot, you can use bone marrow lysate.
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 alongside the samples.
⚠️Feasibility: HCK associates with membranes and lysosomes, so extraction conditions may affect its signal.
HPA tissue expression evidence for HCK
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 | Medium | Protein (IHC) | HPA → |
| Lung | macrophages | Medium | Protein (IHC) | HPA → |
| Testis | pachytene spermatocytes | 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 HCK Western Blot Tips
Deeper troubleshooting and optimisation questions for HCK, answered from its protein features.
How should HCK 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.
Which HCK isoforms could change band size?
Isoforms · UniProt lists four isoforms. Isoforms 2 and 3 lack residues 1–21; isoforms 3 and 4 lack residue 76. These sequence differences may affect migration, but they do not establish where each isoform will appear on a blot.
Check the antibody epitope against the isoform sequences. An epitope within residues 1–21 would be absent from isoforms 2 and 3; one dependent on residue 76 may miss isoforms 3 and 4. These coordinates use the supplied UniProt canonical sequence.
Check the antibody epitope against the isoform sequences. An epitope within residues 1–21 would be absent from isoforms 2 and 3; one dependent on residue 76 may miss isoforms 3 and 4. These coordinates use the supplied UniProt canonical sequence.
Could phosphorylation cause an HCK band shift?
PTM · HCK has seven annotated phosphorylation sites, but their presence alone does not establish a visible band shift. Compare matched samples if testing whether a shifted band tracks with phosphorylation.
In the supplied UniProt coordinates, HCK has phosphothreonine at 36 and 202, phosphoserine at 462, and phosphotyrosine at 51, 209, 411 and 522. Phosphorylation at 51 and 411 is annotated as autocatalytic. Check an antibody’s numbering convention before matching its stated site to these coordinates.
In the supplied UniProt coordinates, HCK has phosphothreonine at 36 and 202, phosphoserine at 462, and phosphotyrosine at 51, 209, 411 and 522. Phosphorylation at 51 and 411 is annotated as autocatalytic. Check an antibody’s numbering convention before matching its stated site to these coordinates.
Does this guide establish induction of HCK?
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 HCK?
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 A01073-3 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 HCK 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.
Why is the HCK band near 60 kDa?
Interpretation · The observed band is approximately 60 kDa, close to HCK’s predicted 59.6 kDa. The supplied features do not establish a cause for any small difference between apparent and predicted mass.
Keep the fraction and preparation consistent between samples. UniProt places isoform 1 in the cytosol and at lipid-anchored membranes, including lysosomal and podosome membranes, so a change in fraction recovery could affect its measured band intensity.
Check whether the antibody detects all four isoforms and whether its epitope overlaps residues 1–21 or 76. HCK also has annotated phosphorylation sites, but those features alone cannot identify an extra band or establish a visible shift.
Keep the fraction and preparation consistent between samples. UniProt places isoform 1 in the cytosol and at lipid-anchored membranes, including lysosomal and podosome membranes, so a change in fraction recovery could affect its measured band intensity.
Check whether the antibody detects all four isoforms and whether its epitope overlaps residues 1–21 or 76. HCK also has annotated phosphorylation sites, but those features alone cannot identify an extra band or establish a visible shift.
Boster reagents
HCK 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-HCK Antibody Picoband®
Cat # A01073-3
A01073-3 is a rabbit polyclonal anti-HCK antibody listed for human reactivity. Its Western blot image shows an approximately 60 kDa band in human THP-1 cells and rat spleen tissue. No publication evidence is supplied.
Which to pick: A01073-3 is the only listed option and has a Western blot image. Consider it for human THP-1 lysate; the image also includes rat spleen, although catalogued reactivity is human only.
Source: BosterBio HCK gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry P08631.
- Human Protein Atlas. HCK tissue expression.
- PMC8435456 — target-verified WB comparison
- PMC1899183 — target-verified WB comparison