Functional Classification and Experimental Applications of Cysteine Endopeptidases: Plant Proteases, Cathepsins, and Arg-Specific Endoproteases

Cysteine endopeptidases are a class of proteases in which a catalytic cysteine residue participates in peptide bond hydrolysis. They include plant-derived proteolytic enzymes, lysosomal cathepsins, and endoproteases with specific cleavage-site preferences. Papain, ficin, and chymopapain are mainly used for protein hydrolysis, tissue digestion, and sample processing; Cathepsin B, Cathepsin L, Cathepsin K, and Cathepsin S are more suitable for studies of lysosomal function, tumor invasion, bone resorption, and inflammatory immunity; Clostripain and Arg-C are commonly used for arginine-site digestion, protein sequencing, and mass spectrometry peptide mapping.

Keywords: cysteine endopeptidase; papain; ficin; cathepsin; Cathepsin B; Cathepsin K; Clostripain; Arg-C

1 Classification Basis of Cysteine Endopeptidases

1.1 Enzymological Positioning of Cysteine Endopeptidases

(1) Catalytic center characteristics

The catalytic activity of cysteine endopeptidases depends on the thiol state of the active-site cysteine residue. This thiol group usually forms a catalytic environment with residues such as histidine, performs nucleophilic attack on substrate peptide bonds, and completes the hydrolysis reaction. Because the active cysteine is susceptible to oxidation, alkylation, or irreversible inhibitors, experimental systems for this class of enzymes usually need to consider pH, reducing agents, metal ions, inhibitors, and sample storage conditions.

(2) Differences in substrate recognition

Cysteine endopeptidases do not represent a uniform substrate specificity. Papain, ficin, and chymopapain have relatively broad proteolytic ranges and are suitable for processing complex protein substrates. Cathepsin B, L, K, and S have defined cellular localization and physiological functional contexts. Clostripain and Arg-C are mainly characterized by their preference for cleavage at arginine-related sites. In experiments, enzymes should not be used interchangeably based only on the shared label of “cysteine protease.”

(3) Differences in research attributes

Plant-derived cysteine proteases are usually used as tool enzymes, mainly serving sample processing, tissue digestion, antigen retrieval, or protein hydrolysis. Cathepsins can serve both as detection targets and as mechanistic indicators for explaining lysosomal function, extracellular matrix degradation, inflammatory responses, and tissue remodeling. Arg-specific endoproteases are more commonly used in protein analysis workflows, emphasizing interpretable cleavage sites and peptide coverage.

1.2 Major Enzyme Types and Functional Grouping

In experimental applications, cysteine endopeptidases can be divided into three groups according to “source–function–application scenario.” The first group consists of plant-derived cysteine proteases, including papain, ficin, and chymopapain, which are mainly used for protein hydrolysis and sample pretreatment. The second group consists of the cathepsin family, including Cathepsin B, L, K, and S, which are mainly used in studies of cellular function and disease mechanisms. The third group consists of specific endoproteases, including Clostripain and Arg-C, which are mainly used for arginine-site digestion and mass spectrometry analysis.

Table 1 Functional grouping of cysteine endopeptidases

Group	Representative enzymes	Functional features	Main applications	Experimental considerations
Plant-derived cysteine proteases	Papain, ficin, chymopapain	Broad substrate range and strong proteolytic capacity	Protein hydrolysis, tissue digestion, antigen retrieval, sample processing	Control enzyme amount, reaction time, pH, reducing environment, and overdigestion
Lysosomal cathepsins	Cathepsin B, L, K, S	Defined cellular localization and functional background	Lysosomal function, tumor invasion, bone resorption, inflammatory immunity	Distinguish expression level, mature form, enzyme activity, and subcellular localization
Arg-specific endoproteases	Clostripain, Arg-C	Strong preference for cleavage at arginine-related sites	Protein sequencing, peptide mapping, supplementary digestion for mass spectrometry	Control reducing conditions, enzyme-to-substrate ratio, digestion time, and missed cleavage rate

2 Plant-Derived Cysteine Proteases

2.1 Papain

(1) Enzymological characteristics

Papain is one of the widely used plant-derived cysteine endopeptidases and is usually obtained from papaya latex. It has a broad proteolytic range and can act on various native and denatured proteins, making it highly applicable in tissue digestion, protein degradation, and antigen retrieval. Papain activity depends on the active cysteine residue. In reaction systems, conditions such as L-cysteine, DTT, or EDTA are often used to maintain enzyme activity, but the concentration of reducing agents and the buffer system must be compatible with downstream assays.

(2) Tissue digestion and cell release

Papain can be used for gentle digestion of certain tissue samples, especially in neural tissue, soft tissue, or samples with complex extracellular matrix components. Its advantage is that it can disrupt proteinaceous structures and promote cell release. Its limitation is that overdigestion may affect the integrity of cell-surface antigens, receptors, adhesion molecules, and membrane proteins. If downstream applications include flow cytometry, primary cell culture, or single-cell analysis, cell recovery, cell viability, and retention of key surface markers should be evaluated simultaneously.

(3) Antigen retrieval and protein hydrolysis

In immunohistochemistry or immunofluorescence samples, papain can expose antigen epitopes masked by fixation-induced crosslinking through limited proteolysis. The key in this application is not maximizing digestion intensity, but maintaining tissue structure, reducing background, and improving target epitope accessibility. In protein hydrolysis experiments, papain is suitable for crude hydrolysis, limited hydrolysis, and comparison of protein sensitivity, but it is not suitable as a highly site-specific protease for mass spectrometry digestion.

2.2 Ficin

(1) Substrate hydrolysis characteristics

Ficin is derived from fig latex and is also a plant-derived cysteine protease. It has a broad substrate range and can hydrolyze various protein substrates. It is commonly used for protein degradation, antibody fragmentation, and sample processing. Compared with papain, ficin may differ in cleavage pattern and reaction efficiency on different protein substrates, making it more suitable as a parallel screening enzyme rather than a direct replacement.

(2) Application in antibody fragmentation

Ficin can be used for immunoglobulin fragmentation and studies of protein structural exposure. This application requires optimization of reaction conditions based on antibody isotype, target fragment type, and downstream detection method. If the goal is to obtain specific Fab, Fc, or hinge-region-related fragments, fragment size and integrity should be confirmed by non-reducing/reducing SDS-PAGE, SEC, mass spectrometry, or immunodetection, rather than interpreting total protein reduction as successful formation of the target fragment.

(3) Boundaries of sample processing

Ficin is suitable for comparing proteolytic capacity and for some sample pretreatment workflows. However, because its substrate selectivity is not equivalent to that of mass-spectrometry-grade specific proteases, it should not be used directly for peptide mapping that requires defined cleavage sites. If the experimental goal is to obtain interpretable peptides, trypsin, Lys-C, Glu-C, Clostripain, Arg-C, or other digestion systems more suitable for sequence analysis should be prioritized.

2.3 Chymopapain

(1) Functional positioning

Chymopapain is also derived from the papaya latex system and belongs to the group of plant cysteine proteases with proteolytic activity. Its main applications include matrix hydrolysis, protein degradation, and comparison of tissue processing conditions. Similar to papain, chymopapain is more suitable as a sample processing tool than as a specific endoprotease for precise site analysis.

(2) Tissue and matrix processing

In samples rich in structural proteins, collagen-related components, or complex extracellular matrix, chymopapain can be used to compare the effects of different protease systems on tissue dispersion and matrix hydrolysis. These experiments should simultaneously assess tissue structure preservation, cell viability, protein integrity, and downstream detection signals. If the goal is to obtain viable cells, “more complete digestion” should not be used as the sole evaluation standard.

(3) Combined use with other plant enzymes

Papain, ficin, and chymopapain can be used for condition screening in the same category of samples, but a unified evaluation system should be established. Protein hydrolysis experiments can compare residual protein bands, peptide distribution, and functional activity. Tissue digestion experiments can compare cell recovery, viability, surface antigen retention, and subsequent culture performance. Different applications should not use a single evaluation standard.

3 Functional Differences of the Cathepsin Family

3.1 Cathepsin B

(1) Lysosomal protein degradation

Cathepsin B is a commonly used lysosomal cysteine protease marker involved in intracellular protein degradation, lysosomal protein turnover, and proteostasis maintenance. Increased expression does not necessarily indicate enhanced lysosomal degradation, because precursor protein, mature enzyme, cytosolic release, and extracellular secretion can all affect detection results. In lysosomal function studies, enzyme activity assays, LAMP1/LAMP2, lysosomal pH, LC3, p62, and activity probes should be combined for comprehensive interpretation.

(2) Tumor invasion and matrix degradation

Cathepsin B can participate in extracellular matrix degradation, tumor cell migration, and invasion. In tumor invasion experiments, Cathepsin B can be analyzed together with MMPs, uPA/uPAR, integrins, ECM degradation assays, and Matrigel invasion results. Detection of Cathepsin B total protein or mRNA alone is insufficient to support its direct contribution to the invasive phenotype.

(3) Inflammatory injury and lysosomal membrane permeabilization

In inflammation and cell injury research, Cathepsin B can be associated with lysosomal membrane permeabilization, inflammasome activation, and cell death. If the NLRP3-Caspase-1-IL-1β axis is being studied, lysosomal integrity, Cathepsin B activity, inflammatory factor release, and cell death indicators should be detected simultaneously to distinguish correlative changes from mechanistic involvement.

3.2 Cathepsin L

(1) Protein turnover and cellular remodeling

Cathepsin L has strong endopeptidase activity and participates in protein turnover, tissue remodeling, and cell migration-related processes. It is commonly used to evaluate lysosomal protein degradation, cellular remodeling, and tumor migration-related mechanisms. Compared with Cathepsin B, Cathepsin L places greater emphasis on protein substrate degradation and tissue remodeling, but its functional status still requires confirmation through activity assays and localization analysis.

(2) Role in antigen processing

Cathepsin L can participate in antigen processing and protein cleavage, affecting antigen peptide generation in immune cells and some non-immune cells. If antigen processing or immune responses are being studied, Cathepsin L should be analyzed together with Cathepsin S, MHC II, CD74/Ii degradation, and antigen presentation efficiency. A change in Cathepsin L expression alone cannot fully explain changes in antigen presentation capacity.

3.3 Cathepsin K

(1) Bone resorption-related collagen degradation

Cathepsin K is closely related to osteoclast-mediated bone resorption and collagen matrix degradation and is one of the representative cysteine cathepsins in bone metabolism research. In osteoclast differentiation experiments, Cathepsin K is often analyzed together with TRAP, RANKL, NFATc1, MMP9, resorption pits, and collagen degradation products to build an evidence chain for bone resorption function.

(2) Bone disease and tumor bone metastasis research

In studies of osteoporosis, bone destruction, and tumor bone metastasis, Cathepsin K can be used to evaluate osteoclast-mediated bone matrix degradation. If experiments only show upregulated Cathepsin K expression without changes in bone slice resorption area, TRAP-positive multinucleated cell number, or collagen degradation markers, they cannot sufficiently demonstrate enhanced bone resorption function.

3.4 Cathepsin S

(1) Antigen presentation and the MHC II pathway

Cathepsin S is closely related to the MHC II pathway in antigen-presenting cells and participates in Ii/CD74 degradation and antigen peptide loading. Compared with Cathepsin B and L, Cathepsin S is more appropriately analyzed within frameworks of antigen processing, inflammatory immunity, and autoimmune research.

(2) Inflammatory microenvironment and extracellular matrix degradation

Cathepsin S activity can change in inflamed tissues, tumor microenvironments, and immune cell infiltration regions. Because it is relatively stable in the extracellular environment, Cathepsin S can also participate in extracellular matrix degradation and immune microenvironment remodeling. Experiments should distinguish intracellular lysosomal activity, secreted enzymatic activity, and local tissue expression, avoiding attribution of all signal changes to the same functional process.

Table 2 Functional emphasis of Cathepsin B, L, K, and S

Enzyme	Main functional emphasis	Suitable research directions	Recommended combined indicators
Cathepsin B	Lysosomal protein degradation, tumor invasion, inflammatory injury	Lysosomal function, ECM degradation, inflammasome	LAMP1, LAMP2, NLRP3, IL-1β, MMPs, invasion assays
Cathepsin L	Protein turnover, cellular remodeling, antigen processing	Tissue remodeling, tumor migration, antigen processing	Activity probes, MHC II, CD74, ECM degradation markers
Cathepsin K	Collagen degradation, bone resorption	Osteoclast function, osteoporosis, bone metastasis	TRAP, RANKL, NFATc1, MMP9, resorption pits
Cathepsin S	Antigen presentation, inflammatory immunity, extracellular matrix degradation	APC function, autoimmunity, tumor immunity	MHC II, CD74, inflammatory factors, immune cell infiltration

4 Arg-Specific Endoproteases

4.1 Clostripain

(1) Cleavage characteristics

Clostripain is a cysteine endopeptidase primarily characterized by cleavage at arginine sites and is commonly used for protein sequencing, peptide mapping, and mass spectrometry sample preparation. Its application value lies in providing peptide fragment combinations different from trypsin digestion, thereby improving sequence coverage in specific regions or assisting the analysis of difficult-to-cover regions.

(2) Application in mass spectrometry pretreatment

In LC-MS/MS experiments, Clostripain can be used as a supplementary digestion system for optimizing protein coverage. For proteins where trypsin generates peptides that are too long, too short, or insufficiently cover key regions, Clostripain can provide new cleavage windows. This method is especially suitable for domain analysis, modification-site localization, and mutation-region verification.

(3) Control of reaction conditions

The performance of Clostripain is affected by reducing environment, enzyme-to-substrate ratio, buffer system, and reaction time. Overdigestion increases nonspecific fragments, whereas insufficient digestion reduces sequence coverage. In mass spectrometry samples, compatibility of reducing agents, salt concentration, and termination method with downstream LC-MS also needs to be considered.

4.2 Arg-C

(1) Site preference

Arg-C endoprotease is usually used for enzymatic cleavage at arginine-related sites and can generate peptide maps different from trypsin digestion. Compared with broad-spectrum plant proteases, Arg-C is more suitable for protein analysis with interpretable cleavage sites and is not suitable for tissue digestion or antigen retrieval.

(2) Complementarity with trypsin

Arg-C can be used to supplement protein regions insufficiently covered by trypsin. For target proteins containing key modification sites, mutation sites, domain linker regions, or obvious missed cleavages in trypsin digestion, Arg-C can improve peptide coverage in specific regions. In quantitative mass spectrometry, digestion conditions should be kept consistent across samples, and quality control proteins or standard peptides should be included to monitor digestion efficiency.

5 Main Application Scenarios and Experimental Design

5.1 Protein Hydrolysis and Tissue Digestion

(1) Protein hydrolysis

Plant-derived cysteine proteases can be prioritized for crude protein hydrolysis, limited hydrolysis, and sample viscosity reduction. These experiments should set interpretation indicators according to the research objective. If total protein degradation is the focus, SDS-PAGE or residual protein content can be used for evaluation. If functional fragments are the focus, immunodetection, SEC, or mass spectrometry should be used to confirm fragment characteristics. If biological activity is the focus, changes or retention of function in hydrolysis products should be evaluated.

(2) Tissue digestion

Tissue digestion experiments should balance cell release efficiency and cell quality. Papain and combined enzyme systems can improve tissue dispersion, but cell viability, surface antigen retention, cell aggregate proportion, and subsequent culture status should be assessed simultaneously. For samples intended for flow cytometry, single-cell sequencing, or primary culture, overdigestion may cause more severe result bias than insufficient digestion.

5.2 Antigen Retrieval and Immunodetection

Protease retrieval is suitable for some antigens whose epitopes are masked after fixation. Cysteine proteases such as papain can improve antigen accessibility through limited hydrolysis, but tissue structure damage, increased background, and nonspecific staining may also occur. During immunohistochemistry or immunofluorescence optimization, no retrieval, heat-induced retrieval, and enzyme retrieval conditions should be compared, and evaluation should integrate target signal, background, tissue morphology, and reproducibility.

5.3 Lysosomal Function and Cellular Stress

Cathepsin B and Cathepsin L are commonly used to evaluate lysosomal function. In experiments, expression changes should not be directly interpreted as changes in degradation flux. A more reasonable design is to simultaneously detect Cathepsin activity, lysosomal acidification, LAMP1/LAMP2, LC3-II, p62, and activity probe signals. If lysosomal membrane permeabilization is involved, changes in enzyme distribution within lysosomes, in the cytosol, or in the extracellular space should also be distinguished.

5.4 Tumor Invasion, Bone Resorption, and Inflammatory Immunity

(1) Tumor invasion

Cathepsin B, L, and S can participate in tumor cell migration, matrix degradation, and invasive phenotypes. Experimental design should connect cathepsin changes with functional assays, including Matrigel invasion, ECM degradation, migration assays, inhibitor treatment, and genetic intervention. Detection of cathepsin expression alone is insufficient to prove that it drives the invasion process.

(2) Bone resorption

Cathepsin K is a key enzyme in osteoclast bone resorption research. Bone metabolism experiments should combine TRAP staining, RANKL stimulation, NFATc1 expression, bone slice resorption area, and collagen degradation markers for integrated interpretation. Cathepsin K inhibitor experiments should also measure cell viability and osteoclast differentiation status to avoid misinterpreting cytotoxicity as inhibition of bone resorption.

(3) Inflammatory immunity

Cathepsin S is more suitable for antigen presentation and inflammatory immunity research, while Cathepsin B is often associated with lysosomal injury and inflammasome activation. Both can participate in inflammatory processes, but their downstream mechanisms differ. Antigen presentation studies should focus on MHC II, CD74, and T-cell responses. Inflammasome studies should focus on NLRP3, Caspase-1, IL-1β, and cell death indicators.

Table 3 Enzyme selection for different experimental purposes

Experimental purpose	Preferred enzymes	Key interpretation indicators	Notes
Crude protein hydrolysis	Papain, ficin, chymopapain	Degree of protein degradation, fragment distribution, function retention	Control enzyme amount and time to avoid overhydrolysis
Tissue digestion	Papain and combined enzyme systems	Cell recovery, viability, surface antigen retention	Conditions need optimization for downstream flow cytometry or single-cell experiments
Antigen retrieval	Papain and other enzyme retrieval systems	Target signal, background, tissue morphology	Staining enhancement alone should not be used as the success criterion
Lysosomal function	Cathepsin B, Cathepsin L	Enzyme activity, lysosomal pH, LAMP1/LAMP2, LC3, p62	Total protein expression cannot replace activity assays
Tumor invasion	Cathepsin B, L, S	Invasion assays, ECM degradation, inhibitor validation	A functional evidence chain is required
Bone resorption	Cathepsin K	TRAP, RANKL, NFATc1, resorption pits	Distinguish osteoclast differentiation from bone resorption activity
Inflammatory immunity	Cathepsin S, Cathepsin B	MHC II, CD74, NLRP3, IL-1β	Mechanisms should be distinguished according to antigen presentation or inflammatory injury
Mass spectrometry digestion	Clostripain, Arg-C	Peptide length, missed cleavage rate, sequence coverage	Verify compatibility of digestion conditions with LC-MS

6 Related Reagent and Material Selection

Table 4 Plant-derived cysteine proteases and Arg-specific endoprotease products

Application module	Cat. No.	Product Name	Grade/Specification	Application Positioning
Papain	P123425	Papain	EnzymoPure™, lyophilized powder,≥10 units/mg,with BAEE as substrate	Routine protein hydrolysis, tissue digestion, and condition screening
Papain	P164463	Papain	Bioactive,ActiBioPure™,Native,High Performance,EnzymoPure™,from Carica papaya; ≥2000U/mg enzyme powder, with casein as substrate	High-activity papain system, suitable for protein hydrolysis and sample processing
Papain	P754923	Papain from Carica papaya	solution, light brown,≥10 U/mg protein (~25 mg/ml)	Liquid enzyme preparation, suitable for digestion systems requiring solution-format enzyme source
Papain	P128675	Papain from Carica papaya Latex(Lyophilized)	EnzymoPure™, ≥15 units/mg protein, with BAEE as substrate	Lyophilized papaya latex-derived enzyme, suitable for optimization of tissue and protein processing conditions
Papain	P128674	Papain from Carica papaya Latex(Suspension)	EnzymoPure™, ≥20 units/mg protein, with BAEE as substrate	Suspension-format papaya latex-derived enzyme, suitable for liquid digestion system screening
Papain	P426757	Papain	EnzymoPure™, 10mM in DMSO	Suitable for experiments involving solution stock formats; compatibility with enzyme activity assays or cell experiments should be confirmed before use
Ficin	F755267	Ficin from fig tree latex	saline suspension,≥1.0 units/mg protein (biuret)	Suspension-format ficin, suitable for protein hydrolysis and sample processing
Ficin	F755315	Ficin from fig tree latex	lyophilized powder	Lyophilized ficin, suitable for self-prepared reaction systems and condition screening
Ficin	D304861	Ficin, from fig tree latex	EnzymoPure™, ≥ 100u/mg	High-activity ficin, suitable for optimization of protein hydrolysis and antibody fragmentation conditions
Chymopapain	C1440278	Chymopapain from Papaya	EnzymoPure™,Native,≥40,000 IMCU/g enzyme powder	Matrix hydrolysis, tissue processing, and protein degradation comparison
Clostripain	C279019	Clostripain NB From Clostridium Histolyticum	EnzymoPure™, Native activity: ≥ 5.0 U/mg (BAEE)	Arg-preferential digestion, protein fragmentation, and digestion condition screening
Clostripain	C128677	Clostripain from Clostridium histolyticum	Bioactive,ActiBioPure™,High Performance,EnzymoPure™,Native,≥50 units/mg dry weight	Protein sequencing, peptide mapping, and supplementary digestion for mass spectrometry
Arg-C	E1456326	Endoproteinase Arg-C（MS）	Recombinant, Bioactive, EnzymoPure™, ActiBioPure™, suitable for mass spectrometry (MS), Native, ≥50 U/mg powder	Arginine-site digestion, LC-MS/MS pretreatment, and peptide coverage optimization

Table 5 Cathepsin B/L/K/S protein and enzyme activity-related products

Application module	Cat. No.	Product Name	Grade/Specification	Application Positioning
Cathepsin B protein	np001112	Cathepsin B, Human Liver	Bioactive,ActiBioPure™,Native,High Performance,EnzymoPure™,≥95%(SDS-PAGE),≥200 U/mg protein; Protein concentration: See COA	Natural-source Cathepsin B for enzyme activity, substrate hydrolysis, and inhibitor validation
Cathepsin B protein	rp183609	Recombinant Human Cathepsin B Protein	Animal Free,Carrier Free,Bioactive,ActiBioPure™,His Tag,PBS Only,≥95%(SDS-PAGE)	Human recombinant Cathepsin B for lysosomal function, substrate, and inhibitor studies
Cathepsin B protein	rp153668	Recombinant Mouse Cathepsin B Protein	Animal Free,Carrier Free,Bioactive,ActiBioPure™,His Tag,≥95%(SDS-PAGE)	Mouse-source Cathepsin B research, suitable for animal model-related systems
Cathepsin L protein	np001107	Cathepsin L from Human Liver	Bioactive,ActiBioPure™,Native,High Performance,EnzymoPure™,≥95%(SDS-PAGE),≥1 U/mg protein; Protein concentration: See COA	Natural-source Cathepsin L for protein turnover, substrate hydrolysis, and inhibitor validation
Cathepsin L protein	C1449054	Cathepsin L		Cathepsin L-related enzymology research and detection system development
Cathepsin L protein	rp181235	Recombinant Human Cathepsin L Protein	Animal Free,Carrier Free,His Tag,≥90%(SDS-PAGE),See COA	Human recombinant Cathepsin L for protein degradation and cellular remodeling research
Cathepsin L protein	rp224963	Recombinant Human Cathepsin L Protein	Animal Free,Carrier Free,His Tag,≥90%(SDS-PAGE),See COA	Human Cathepsin L recombinant protein for substrate and inhibitor screening
Cathepsin L protein	rp153675	Recombinant Mouse Cathepsin L Protein	Animal Free,Carrier Free,Bioactive,ActiBioPure™,High Performance,His Tag,≥95%(SDS-PAGE)	Mouse Cathepsin L-related enzyme activity and mechanism research
Cathepsin K protein	C1444277	Cathepsin K		Bone resorption, collagen degradation, and Cathepsin K-related functional research
Cathepsin S protein	C1443361	Cathepsin S, human		Cathepsin S-related enzymology research and antigen processing mechanism research
Cathepsin S protein	rp181600	Recombinant Human Cathepsin S Protein	Animal Free,Carrier Free,Bioactive,ActiBioPure™,His Tag,PBS Only,≥90%(SDS-PAGE)	Human recombinant Cathepsin S for antigen presentation, inflammatory immunity, and substrate validation
Cathepsin S protein	rp143673	Recombinant Human Cathepsin S protein	Animal Free,Carrier Free,Bioactive,ActiBioPure™,Azide Free,His Tag,≥90%(SDS-PAGE),See COA	Cathepsin S activity detection, inhibitor screening, and immune-related mechanism research

Table 6 Cathepsin B/L/K/S antibody and ELISA detection products

Application module	Cat. No.	Product Name	Grade/Specification	Application Positioning
Cathepsin B antibody	Ab093155	Cathepsin B Mouse mAb	Carrier Free, ExactAb™, Azide Free, Validated, High Performance, See COA	Cathepsin B protein detection, suitable for WB, IF, or related immunodetection
Cathepsin B antibody	Ab325784	Recombinant Cathepsin B Antibody	KD Validation	Specific Cathepsin B detection, suitable for experiments requiring knockdown validation support
Cathepsin B antibody	Ab001150	Anti-Cathepsin B Polyclonal Ab produced in rabbit	Protein Concentration: 4.24 mg/mL	Cathepsin B expression detection and tissue sample analysis
Cathepsin B ELISA	EJ1514552	Human Cathepsin B (CTSB) ELISA Kit	BioReagent	Detection of Cathepsin B content in human samples
Cathepsin B ELISA	H1509933	Human pro-Cathepsin B ELISA Kit	BioReagent	Detection of pro-Cathepsin B in human samples, used to distinguish precursor protein levels
Cathepsin B ELISA	H1509937	Human Cathepsin B ELISA Kit	BioReagent	Quantitative detection of Cathepsin B in human samples
Cathepsin B ELISA	EJ1512216	Rat Cathepsin B (CTSB) ELISA Kit	BioReagent	Detection of Cathepsin B in rat samples
Cathepsin B ELISA	EJ1513029	Mouse Cathepsin B (CTSB) ELISA Kit	BioReagent	Detection of Cathepsin B in mouse samples
Cathepsin L antibody	Ab093200	Cathepsin L/MEP Antibody	ExactAb™, Validated, 1.0 mg/mL	Cathepsin L/MEP-related protein detection
Cathepsin L antibody	Ab001147	Anti-Cathepsin L Polyclonal Ab produced in rabbit	Protein Concentration: 9.15 mg/mL	Cathepsin L expression detection and tissue sample analysis
Cathepsin L/K-related antibody	Ab093201	Recombinant Cathepsin L/V/K/H Antibody	ExactAb™, Validated, Recombinant, 0.5 mg/mL	Cathepsin L/V/K/H-related detection, suitable for experiments with defined cross-recognition needs
Cathepsin L/K-related antibody	Ab326677	Recombinant Cathepsin L/V/K/H Antibody	KD Validation	Cathepsin L/V/K/H-related detection, suitable for experiments requiring knockdown validation support
Cathepsin L/K-related antibody	Ab331049	Recombinant Cathepsin L/V/K/H Antibody	Recombinant,ExactAb™,Validated,PBS Only,See COA	Cathepsin L/V/K/H protein detection; target distinction should be considered during use
Cathepsin L ELISA	EJ1514557	Human Cathepsin L (Cath-L) ELISA Kit	BioReagent	Detection of Cathepsin L in human samples
Cathepsin L ELISA	H1509932	Human CTSL/Cathepsin L ELISA Kit	BioReagent	Quantitative detection of Cathepsin L in human samples
Cathepsin L ELISA	EJ1512219	Rat Cathepsin L (CTSL) ELISA Kit	BioReagent	Detection of Cathepsin L in rat samples
Cathepsin L ELISA	EJ1513034	Mouse Cathepsin L (CTSL) ELISA Kit	BioReagent	Detection of Cathepsin L in mouse samples
Cathepsin K ELISA	EJ1514556	Human Cathepsin K (Cath-K) ELISA Kit	BioReagent	Detection of Cathepsin K in human samples, suitable for bone resorption-related research
Cathepsin K ELISA	EJ1512218	Rat Cathepsin K (Cath-K) ELISA Kit	BioReagent	Detection of Cathepsin K in rat samples
Cathepsin K ELISA	EJ1513033	Mouse Cathepsin K (CTSK) ELISA Kit	BioReagent	Detection of Cathepsin K in mouse samples
Cathepsin S antibody	Ab093204	Cathepsin S Mouse mAb	Carrier Free, ExactAb™, Azide Free, Validated, High Performance, See COA	Cathepsin S expression detection, suitable for antigen presentation and inflammatory immunity research
Cathepsin S antibody	Ab191933	Fsn0503h (anti-Cathepsin S)	Carrier Free, Recombinant, ExactAb™, Low Endotoxin, Azide Free, Validated, Animal Free, ≥95%(SDS-PAGE&SEC-HPLC), See COA	Cathepsin S-specific antibody, suitable for highly consistent immunodetection
Cathepsin S ELISA	EJ1514558	Human Cathepsin S (Cath-S) ELISA Kit	BioReagent	Detection of Cathepsin S in human samples
Cathepsin S ELISA	H1509923	Human Cathepsin S ELISA Kit	BioReagent	Quantitative detection of Cathepsin S in human samples
Cathepsin S ELISA	EJ1512220	Rat Cathepsin S (Cath-S) ELISA Kit	BioReagent	Detection of Cathepsin S in rat samples
Cathepsin S ELISA	EJ1513035	Mouse Cathepsin S (Cath-S) ELISA Kit	BioReagent	Detection of Cathepsin S in mouse samples

7 Key Issues in Result Interpretation

7.1 Distinguishing Expression Level from Enzyme Activity

Cathepsins are often synthesized as precursor forms and acquire activity after processing and maturation. mRNA upregulation, increased total protein, or enhanced ELISA signal only indicates changes in expression or content and cannot directly represent enhanced enzymatic activity. In mechanistic studies, activity substrates, activity probes, specific inhibitors, or genetic interventions should be included whenever possible.

7.2 Matching Digestion Intensity to Experimental Purpose

Plant-derived proteases have strong hydrolytic capacity, but overdigestion can cause antigen loss, cell damage, or destruction of target fragments. In tissue digestion and antigen retrieval experiments, time gradients and enzyme concentration gradients should be established first. In protein hydrolysis experiments, reaction suitability should be judged according to the target product rather than total degradation intensity.

7.3 Evidence Level of Inhibitor Experiments

Broad-spectrum Cathepsin inhibitors, Cathepsin L inhibitors, Cathepsin K inhibitors, and Cathepsin S inhibitors can be used to validate protease dependence, but inhibitor experiments need to be interpreted together with cell viability, off-target effects, and dose-response relationships. When studying a specific enzyme, selective inhibitors, siRNA/CRISPR intervention, recombinant enzyme, or rescue experiments are recommended to improve the reliability of mechanistic conclusions.

Experimental selection of cysteine endopeptidases should be based on functional positioning: plant-derived proteases are suitable for protein hydrolysis and sample processing; Cathepsin B/L/K/S are suitable for studies of cellular function and disease mechanisms; Clostripain and Arg-C are suitable for site-preferential digestion and mass spectrometry analysis. In practical applications, enzyme activity, localization, substrate, inhibitors, and functional experiments should be integrated to avoid interpretation based only on enzyme names or expression levels.