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- Table of Contents
CTSH / Pro-cathepsin H · Western blot design guide
Design a Western Blot for CTSH
Source-linked CTSH Western blot protocol options, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-CTSH 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
CTSH 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 | ~37.4 kDa | |
| Observed band | ≈28 kDa | |
| Gel | 12% (catalog M01510) | |
| Positive control | Appendix (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 | Proteolytic maturation | |
| Gene-set association | MSigDB Hallmark membership | |
| Isoform | 1 isoform(s) |
Section 1
Source-Linked CTSH Western Blot Protocol Options
The M01510 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | human RT4, human A431 (catalog M01510) |
| Gel % | 12% (catalog M01510) |
| Load | 30 ug; reducing conditions (catalog M01510) |
| Transfer | a nitrocellulose membrane at 150 mA for 50-90 minutes (catalog M01510) |
| Membrane | nitrocellulose membrane (catalog M01510) |
| Blocking | 5% non-fat milk/TBS for 1.5 hour at RT (catalog M01510) |
| Primary antibody | M01510 · 1:500 (catalog M01510) |
| Primary incubation | overnight at 4°C (catalog M01510) |
| Secondary antibody | goat anti-rabbit IgG-HRP, 1:5000 (catalog M01510) |
| Secondary incubation | 1.5 hour at RT (catalog M01510) |
| Wash | TBS-0.1%Tween 3 times with 5 minutes each (catalog M01510) |
| Detection | ECL (catalog M01510) |
Section 2
What Is the Expected CTSH Western Blot Band Size?
CTSH is predicted at 37.4 kDa, but an approximately 28 kDa band is observed; the cause of that difference is not established.
| Band at approximately 28 kDa | Empirical CTSH band in reducing whole-cell lysates; its molecular form is not established |
| Band near 37.4 kDa | Possible full-length pro-cathepsin H precursor |
| Band below the precursor position | Possible processed CTSH containing fewer residues |
| Several bands at different positions | Possible precursor and processed forms, including disulfide-linked chains that separate upon reduction |
| Band migrating above a corresponding unglycosylated form | Possible contribution from N-linked glycosylation at Asn101 or Asn230 |
💡Expected CTSH appearanceThe predicted full-length precursor is 37.4 kDa, while reducing whole-cell blots show approximately 28 kDa; the molecular form behind that difference needs confirmation with band-identity controls.
| Predicted full-length precursor mass | 37.4 kDa by sequence; the empirical band is approximately 28 kDa |
| N-linked glycosylation at Asn101 | May increase apparent size if occupied; its visible effect is not established |
| N-linked glycosylation at Asn230 | May increase apparent size if occupied; its visible effect is not established |
| Signal peptide at residues 1–22 | Cleavage removes residues from the precursor, but its band shift is not established |
| Propeptide at residues 23–97 | Cleavage removes residues during maturation, but its contribution to the 28 kDa band is not established |
| Disulfide-linked mini and large chains | Reduction can separate processed chains; no fragment masses are supplied |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | CTSH is lysosomal and may be scarce in the sampled lysate | Check a lysosome-enriched fraction and a positive-control lysate |
| Band higher than expected | Possible N-linked glycosylation or incompletely reduced disulfide-linked chains | Compare deglycosylated and fully reduced aliquots |
| Band lower than expected | CTSH processing may remove its signal peptide and propeptide or separate its chains | Compare with the approximately 28 kDa reference band and verify identity with an independent antibody |
| Broad smear instead of sharp band | Variable occupancy or composition at the two N-linked glycosylation sites is possible | Compare an enzymatically deglycosylated aliquot with an untreated control |
| Multiple bands | Precursor and processed CTSH forms or chain separation may contribute | Compare reducing conditions and antibodies recognizing different CTSH regions |
| Fragments below expected size | The large chain may split into heavy and light chains | Check reducing conditions and antibody epitope coverage |
Experiment evidence
Western blot images showing CTSH band
M01510 · CTSH
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for CTSH Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for CTSH in Western blot, you can use appendix tissue, which HPA scores as high for CTSH.
Positive control: Appendix (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: HPA identifies adipose tissue as not detected, but verify its negative signal in your blot.
HPA tissue expression evidence for CTSH
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 | germinal center cells | High | Protein (IHC) | HPA → |
| Cervix | glandular cells | High | Protein (IHC) | HPA → |
| Colon | mucosal lymphoid cells | High | Protein (IHC) | HPA → |
| Duodenum | endocrine cells | High | Protein (IHC) | HPA → |
| Gallbladder | glandular cells | 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 → |
| Caudate | glial cells | Not detected | Protein (IHC) | HPA → |
| Cerebellum | cells in granular layer | Not detected | Protein (IHC) | HPA → |
| Cerebral cortex | endothelial cells | Not detected | Protein (IHC) | HPA → |
| Esophagus | squamous epithelial cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced CTSH Western Blot Tips
Deeper troubleshooting and optimisation questions for CTSH, answered from its protein features.
How should CTSH 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.
Are multiple CTSH isoforms expected?
Isoforms · The supplied UniProt record lists one isoform and no alternative sequence. If several bands appear, consider processing and chain composition before assigning them to isoforms.
Could glycosylation change CTSH band mobility?
PTM · UniProt lists N-linked glycosylation at Asn101 and Asn230, using its supplied residue numbering. These sites could affect mobility, but their presence alone does not demonstrate a visible shift. Compare samples under the same preparation conditions.
Does this guide establish induction of CTSH?
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 CTSH?
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 M01510 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.
Which CTSH band should be quantified across samples?
Quantitation · Choose a consistent band or defined set of bands across samples, and state whether the measurement targets pro-cathepsin H or a processed form. The record describes processing and multiple chains, so combining bands without a defined rule can obscure changes in processing.
Why might CTSH run near 28 kDa instead of 37.4 kDa?
Interpretation · The 37.4 kDa prediction refers to pro-cathepsin H. UniProt lists a signal peptide at residues 1..22 and a propeptide at 23..97, so processing can change the mass detected. The ≈28 kDa band is an observed apparent mass; these features alone do not establish its exact chain identity or fully explain the difference.
UniProt describes a mini chain and a large chain that may split into heavy and light chains. Four disulfide bonds are listed, and the chains are held together by disulfide bonds. Interpret band patterns in light of the sample’s reducing conditions; the features do not specify the mass of each chain.
CTSH is a lysosomal zymogen with a signal peptide at 1..22, a propeptide at 23..97, and a multichain mature form. Different processing states or chains are plausible interpretations. The supplied features cannot identify an unexpected band by size alone.
UniProt describes a mini chain and a large chain that may split into heavy and light chains. Four disulfide bonds are listed, and the chains are held together by disulfide bonds. Interpret band patterns in light of the sample’s reducing conditions; the features do not specify the mass of each chain.
CTSH is a lysosomal zymogen with a signal peptide at 1..22, a propeptide at 23..97, and a multichain mature form. Different processing states or chains are plausible interpretations. The supplied features cannot identify an unexpected band by size alone.
Boster reagents
CTSH 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-Cathepsin H Rabbit Monoclonal Antibody
Cat # M01510
M01510 is a rabbit monoclonal anti-CTSH antibody listed for human reactivity. Its supplied Western blot image uses reducing human RT4 and A431 whole-cell lysates and reports a band near 28 kDa, versus an expected 37 kDa.
Which to pick: M01510 is the only listed option and has a Western blot image for human RT4 and A431 lysates at 1:500. Validation in other samples or species is not documented here; consider the reported 28 versus 37 kDa band discrepancy.
Source: BosterBio CTSH gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry P09668.
- Human Protein Atlas. CTSH tissue expression.
- PMC3192174 — target-verified WB comparison
- PMC11370762 — target-verified WB comparison
- PMC13182238 — target-verified WB comparison