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CTSK · Western blot design guide
Design a Western Blot for CTSK
Real validated CTSK Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-CTSK WB antibodies. Everything you need to plan the experiment before you commit precious samples.
Last reviewed: May 2026 · Scientific review: Boster Bio technical team
CTSK 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 | ~37 kDa | |
| Observed band | 45 kDa | |
| Gel | 10–12% | |
| Negative control | siRNA / KO lysate |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Glycosylated + Cleaved | |
| Caveat | Propeptide cleavage state | |
| Regulation | Coagulation | |
| Isoform | 1 isoform(s) |
Section 1
Real Curated CTSK Western Blot Protocols
Literature-validated Western blot parameters for CTSK — gel percentage, transfer, blocking, antibody incubation and detection, extracted from published methods.
| Sample / lysate | HELA , Lane 2: 22RV1 . After electrophoresis, proteins were transferred to a nitrocellulose membrane at 150 mA for 50-90 minutes. Blocked the membrane with 5% non-fat milk/TBS for 1.5 hour at RT. The membrane was incubated with rabbit anti-Cathepsin K antigen affinity purified polyclonal antibody (Catalog # PB9856) at 0.5 μg/mL overnight at 4°C, then washed with TBS-0.1%Tween 3 times with 5 minutes each and probed with a goat anti-rabbit IgG-HRP secondary antibody at a dilution of 1:5000 for 1.5 hour at RT. The signal is developed using an Enhanced Chemiluminescent detection (ECL) kit (Catalog # EK1002) with Tanon 5200 system. A specific band was detected for Cathepsin K at approximately 45 kDa. The expected band size for Cathepsin K is at 40 kDa |
| Gel % | 10–12% |
| Load | 30 ug |
| Transfer | nitrocellulose membrane, 150 mA, 50–90 min |
| Membrane | nitrocellulose |
| Blocking | 5% non-fat milk / TBS, 1.5 h RT |
| Primary antibody | 0.5 µg/mL |
| Primary incubation | overnight at 4 °C |
| Secondary antibody | goat anti-rabbit IgG-HRP, 1:5000 |
| Wash | TBS-0.1% Tween, 3 × 5 min |
| Detection | ECL |
| Exposure / imaging | Tanon 5200 |
| Observed band | 45 kDa |
Section 2
What Is the Expected CTSK Western Blot Band Size?
CTSK predicts a 37 kDa core mass but runs at 45 kDa on blots, the glycosylated proenzyme, due to N-glycosylation at Asn103 rather than mature processed enzyme.
| Predominant band around 45 kDa | glycosylated cathepsin K proenzyme, running above the 37 kDa core mass predicted from sequence |
| Band near 37 kDa or slightly lower | unmodified or minimally glycosylated core polypeptide close to the predicted mass |
| Band well below the 37-45 kDa range | mature cathepsin K generated after removal of the signal peptide and the 16-114 propeptide |
| Diffuse or smeared band rather than one sharp line | heterogeneous glycan occupancy at the single N-linked site on Asn103 |
| Faint or absent signal in whole-cell lysate despite good sample loading | a portion of cathepsin K is secreted or membrane-associated rather than retained intracellularly |
💡Expected CTSK appearanceExpect a predominant band at 45 kDa, the glycosylated cathepsin K proenzyme, rather than the 37 kDa core mass predicted directly from the unmodified polypeptide sequence.
| Predicted mass from UniProt | core cathepsin K polypeptide predicts a 37 kDa band before any modification |
| N-glycosylation at Asn103 | adds carbohydrate mass and heterogeneity, shifting and broadening the band up toward the observed 45 kDa |
| Signal peptide (residues 1-15) and propeptide (residues 16-114) cleavage | removal during maturation yields a smaller mature-enzyme band well below the 45 kDa proenzyme species |
| Intrachain disulfide bonds (3 total) | stabilize the folded protease domain and can cause a modest mobility shift between reducing and non-reducing conditions without creating a dimer |
| Bacterial or non-glycosylated recombinant standard | lacking the Asn103 glycan, runs closer to the 37 kDa predicted mass, lower than the native 45 kDa lysate band |
| Situation | Likely cause | Next action |
|---|---|---|
| Band higher than expected | the glycosylated proenzyme form is the native species and already runs at 45 kDa, above the bare 37 kDa prediction | compare against the 45 kDa empirical reference before assuming the higher band is nonspecific |
| Band lower than expected | propeptide has been removed, producing mature processed cathepsin K below the 45 kDa proenzyme size | confirm with a mature-enzyme standard or antibody epitope location to distinguish processed from proform |
| Broad smear instead of sharp band | heterogeneous glycan occupancy at the single N103 site produces a spread of glycoforms | treat lysate with a deglycosylation enzyme to collapse the smear into one defined species |
| No band in lysate | cathepsin K is secreted and membrane-associated as well as lysosomal, so intracellular pools may be low in some cell types | test lysosome-rich or secreting cell lines and consider probing concentrated conditioned media |
| Multiple bands | co-detection of the glycosylated proenzyme alongside partially processed or mature cleavage products | extend gel run time to resolve species and confirm identity against propeptide-region epitope mapping |
| Weak or no signal | cathepsin K expression is enriched in specific cell types, so low native expression can limit detection | verify the sample source expresses cathepsin K before troubleshooting antibody or transfer conditions |
Experiment evidence
Western blot images showing CTSK band
PB9856 · CTSK
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for CTSK Western blot
🧪For positive controls for CTSK in Western blot, you can use thyroid tissue lysate, consistent with UniProt evidence that CTSK localizes to the lumen of thyroid follicles.
Positive control: Thyroid tissue
Negative control: ubiquitously expressed; use siRNA knockdown or KO line
Loading controls: Run GAPDH and β-actin as standard loading controls alongside a total-protein stain such as stain-free imaging, Ponceau S, or REVERT.
⚠️Feasibility: CTSK is secreted and lysosomal/apical-membrane localized with no HPA tissue-expression data available, so whole-cell lysate signal may be weak and no clean negative tissue can be identified — use conditioned medium alongside siRNA knockdown or a KO line to confirm specificity.
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.
Positive expression · recommended positive controls
| Tissue | Cell type | Level | Evidence | Source |
|---|
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|
Section 3
Advanced CTSK Western Blot Tips
Deeper troubleshooting and optimisation questions for CTSK, answered from its protein features.
Why does CTSK run at 45 kDa versus predicted 37 kDa?
CTSK is synthesized as a preproenzyme; the 15-residue signal peptide and 99-residue propeptide (16-114) are cleaved during maturation, but incompletely processed or proenzyme forms retain extra mass. A single N-glycosylation site adds further mass, so unprocessed/glycosylated pro-cathepsin K migrates near 45 kDa rather than the 37 kDa mature-enzyme prediction. Expect multiple bands representing pro- and mature forms.
Does glycosylation affect CTSK Western blot mobility?
CTSK carries one N-glycosylation site, which increases apparent molecular weight and can broaden or shift the band relative to the unmodified polypeptide. This contributes to the observed 45 kDa band running higher than the 37 kDa predicted mass. Glycosidase treatment (e.g., PNGase F) can be used to confirm the contribution of this modification if band identity is uncertain.
How should blocking be optimized for CTSK detection?
Because CTSK is a glycoprotein, milk-based blockers containing glycoproteins can increase nonspecific lectin-like binding; BSA is preferable for blocking and antibody dilution. Standard 5% BSA in TBST for one hour is sufficient given no unusual PTMs beyond glycosylation are annotated.
What transfer method to use for CTSK Western blot?
CTSK is a peripheral apical membrane and secreted/lysosomal protein of modest size (37-45 kDa), so standard wet or semi-dry transfer onto PVDF or nitrocellulose is effective. No large hydrophobic transmembrane domain is annotated, so extended transfer times used for polytopic membrane proteins are not required.
How to normalize CTSK signal for quantitation?
Because CTSK localizes to lysosomes, apical membrane, and secreted fractions rather than uniformly across the cytoplasm, normalize to total protein stain rather than a single cytoplasmic housekeeping gene, especially when comparing whole-cell versus secreted/conditioned-medium samples.
Why might multiple CTSK bands appear on blots?
CTSK is produced as a zymogen: an unprocessed preproenzyme, a proenzyme after signal peptide removal, and a mature enzyme after propeptide (16-114) cleavage. These processing intermediates, plus the glycosylated proenzyme running near 45 kDa, explain multiple bands. Disease variants (annotated as disease-associated) may also alter processing and produce aberrant sizes.
Should CTSK samples be run reduced or non-reduced?
CTSK contains three disulfide bonds that stabilize its mature fold; reducing sample buffer (with DTT or beta-mercaptoethanol) is recommended to linearize the protein for consistent SDS-PAGE migration, since non-reduced samples may show altered mobility or aggregate-associated bands.
Boster reagents
Best CTSK Western Blot Antibodies
BosterBio's CTSK antibodies are among the best-performing WB antibodies on the market — well cited, thoroughly validated, and orthogonally cross-validated against negative tissues and complementary methods.
Real WB data
Anti-Cathepsin K/CTSK Antibody Picoband®
Cat # PB9856
For CTSK Western blots, we recommend the Boster antibody with a documented, gel-verified WB validation image (PB9856), giving direct visual confirmation of specific band detection under standard SDS-PAGE conditions.
Which to pick: Only one Boster CTSK antibody is catalogued here: PB9856. It includes an actual Western blot validation image showing specific detection, so it's the clear default choice — no alternative SKUs are available to compare against.
Source: BosterBio CTSK gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry P43235.
- Human Protein Atlas. CTSK tissue expression.