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- Table of Contents
CTSS / Cathepsin S · Western blot design guide
Design a Western Blot for CTSS
Real validated CTSS Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-CTSS 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
CTSS 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 | ~37.5 kDa | |
| Observed band | ~25 kDa | |
| Gel | 10% (catalog A02514-2) | |
| Positive control | Bone marrow (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 + Cleaved | |
| Caveat | Maturation-state controls | |
| Gene-set association | MSigDB Hallmark membership | |
| Isoform | 2 isoform(s) |
Section 1
Real Curated CTSS Western Blot Protocols
The A02514-2 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | human THP-1 (catalog A02514-2) |
| Gel % | 10% (catalog A02514-2) |
| Load | 30 ug; reducing conditions (catalog A02514-2) |
| Transfer | a nitrocellulose membrane at 150 mA for 50-90 minutes (catalog A02514-2) |
| Membrane | nitrocellulose membrane (catalog A02514-2) |
| Blocking | 5% non-fat milk/TBS for 1.5 hour at RT (catalog A02514-2) |
| Primary antibody | A02514-2 · 0.5 μg/mL (catalog A02514-2) |
| Primary incubation | overnight at 4°C (catalog A02514-2) |
| Secondary antibody | goat anti-rabbit IgG-HRP, 1:5000 (catalog A02514-2) |
| Secondary incubation | 1.5 hour at RT (catalog A02514-2) |
| Wash | TBS-0.1%Tween 3 times with 5 minutes each (catalog A02514-2) |
| Detection | ECL (catalog A02514-2) |
Section 2
What Is the Expected CTSS Western Blot Band Size?
CTSS has a predicted full-length mass of 37.5 kDa and an empirical ~25 kDa band; the cause of their difference is not established.
| Band at ~25 kDa | Empirical CTSS band in reducing THP-1 lysate; confirm identity with antibody controls. |
| Band near 37.5 kDa | Possible full-length precursor near the predicted mass, without empirical confirmation here. |
| Band between ~25 and 37.5 kDa | Processing of the signal peptide or propeptide is possible; its migration is unmeasured. |
| Multiple discrete bands | Precursor processing or isoforms 1 and 2 are possible, but distinct isoform bands are unproven. |
| Little or no band in whole-cell lysate | CTSS can be secreted or localized to lysosomes and phagosomes. |
💡Expected CTSS appearanceThe predicted full-length mass is 37.5 kDa, while a reducing THP-1 blot shows ~25 kDa; the cause of that difference is unestablished, so confirm band identity with antibody controls.
| Predicted full-length mass | 37.5 kDa is calculated; the empirical band is ~25 kDa, with the difference unexplained. |
| Signal peptide at residues 1–16 | Its removal can reduce mass relative to the precursor; resulting migration is unmeasured. |
| Propeptide at residues 17–114 | Its removal can reduce mass relative to the precursor; resulting migration is unmeasured. |
| N-linked glycosylation site at Asn104 | This propeptide site may modify a precursor; a visible shift is not established. |
| Splice isoforms 1 and 2 | They may differ in size, but their relative masses and band separation are unknown. |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | CTSS may be secreted or concentrated in lysosomes or phagosomes. | Check conditioned medium or enriched fractions alongside a positive lysate control. |
| Band higher than expected | An uncleaved precursor could migrate above the empirical ~25 kDa band. | Compare with the ~25 kDa positive control and verify identity with a second antibody or CTSS depletion. |
| Band lower than expected | Further proteolysis may yield material below the empirical ~25 kDa band. | Use protease inhibitors during preparation and verify identity with CTSS depletion. |
| Multiple bands | Precursor processing or isoforms 1 and 2 may contribute; band assignments are unproven. | Compare with a positive control and test which bands disappear after CTSS depletion. |
| Weak or no signal | CTSS distribution between cellular compartments and secreted material may limit the sampled fraction. | Check sample fraction, loading, and a CTSS-positive control. |
Experiment evidence
Western blot images showing CTSS band
A02514-2 · CTSS
M02514 · CTSS
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for CTSS Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for CTSS in Western blot, you can use bone marrow tissue, which HPA rates High.
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.
⚠️Feasibility: CTSS is also secreted, so consider conditioned medium if lysate signal is weak.
HPA tissue expression evidence for CTSS
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 → |
| Cerebral cortex | neuropil | High | Protein (IHC) | HPA → |
| Colon | endocrine cells | High | Protein (IHC) | HPA → |
| Duodenum | endocrine cells | High | Protein (IHC) | HPA → |
| Fallopian tube | ciliated cells (cell body) | 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 → |
| Adrenal gland | glandular cells | Not detected | Protein (IHC) | HPA → |
| Breast | glandular cells | Not detected | Protein (IHC) | HPA → |
| Caudate | glial cells | Not detected | Protein (IHC) | HPA → |
| Cerebellum | cells in granular layer | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced CTSS Western Blot Tips
Deeper troubleshooting and optimisation questions for CTSS, answered from its protein features.
How should CTSS 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.
How can isoform 2 affect a CTSS Western blot?
Isoforms · Isoform 2 lacks residues 84–133 in UniProt isoform 1 numbering. This deletion spans part of the propeptide and includes the listed glycosylation site at Asn104. Compare bands cautiously: the sequence difference does not establish which isoform produces any particular band.
Could glycosylation change CTSS band migration?
PTM · UniProt lists one N-linked site at Asn104, using the supplied full-length sequence numbering. Because Asn104 lies within the propeptide (17–114), a processed species may lack it. The site alone does not demonstrate a visible mobility shift.
Does this guide establish induction of CTSS?
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 CTSS?
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 A02514-2 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 CTSS bands be quantified across samples?
Quantitation · Measure the ~25 kDa band consistently across lanes and report higher bands separately. CTSS has precursor features and two isoforms, so combining distinct bands could obscure changes in the species being measured.
Why is CTSS near 25 kDa instead of predicted 37.5 kDa?
Interpretation · The 37.5 kDa prediction includes the signal peptide (1–16) and propeptide (17–114). Their removal could yield a smaller species. The observed ~25 kDa band is consistent with processing, but these features alone do not establish its identity or explain the exact mass difference.
Consider precursor and processed forms, the two isoforms, and glycosylation at Asn104. CTSS is listed as a zymogen with a signal peptide and propeptide, but band position alone cannot distinguish these possibilities.
UniProt places CTSS in lysosomes and phagosomes and also lists it as secreted. Specify which sample fraction was measured when comparing signals; the listed locations do not predict a particular band size in either fraction.
UniProt lists four disulfide bonds and describes CTSS as a monomer. Keep sample preparation conditions consistent across lanes when comparing migration. The bonds alone do not establish a specific band shift.
Consider precursor and processed forms, the two isoforms, and glycosylation at Asn104. CTSS is listed as a zymogen with a signal peptide and propeptide, but band position alone cannot distinguish these possibilities.
UniProt places CTSS in lysosomes and phagosomes and also lists it as secreted. Specify which sample fraction was measured when comparing signals; the listed locations do not predict a particular band size in either fraction.
UniProt lists four disulfide bonds and describes CTSS as a monomer. Keep sample preparation conditions consistent across lanes when comparing migration. The bonds alone do not establish a specific band shift.
Boster reagents
CTSS 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-CTSS Antibody Picoband®
Cat # A02514-2
Real WB data
Anti-Cathepsin S CTSS Monoclonal Antibody
Cat # M02514
Both listed anti-CTSS antibodies are reported for human samples and have Western blot images: A02514-2 in THP-1 lysate and M02514 in U87-MG lysate. A02514-2 shows a band near 25 kDa despite an expected size of 37 kDa; no independent validation is supplied.
Which to pick: For a THP-1 workflow, A02514-2 provides detailed blot conditions. For U87-MG lysate, M02514 has an image, but its caption gives few experimental details. Both list human reactivity; choose based on the sample and available protocol detail.
Source: BosterBio CTSS gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry P25774.
- Human Protein Atlas. CTSS tissue expression.
- PMC5361207 — target-verified WB comparison
- PMC10728302 — target-verified WB comparison
- PMC12343434 — target-verified WB comparison