Decorin Antibody (Rabbit mAb) [E13A13]

Application: Reactivity: Mouse, Rat, Human

Lane 1: Mouse uterus

Experiment Essentials

WB
Recommended wet transfer conditions: 200 mA, 60 min.

Usage Information

Dilution
WB1:1000 IHC1:2000 FCM1:50

Application
WB, IHC, FCM

Reactivity
Mouse, Rat, Human

Source
Rabbit Monoclonal Antibody

Storage Buffer
PBS, pH 7.2+50% Glycerol+0.05% BSA+0.01% NaN3

Storage (from the date of receipt)
-20°C (avoid freeze-thaw cycles), 2 years

Predicted MW Observed MW
40 kDa 90-140 kDa
^*Why do the predicted and actual molecular weights differ? The following reasons may explain differences between the predicted and actual protein molecular weight. Post-translational modifications(e.g., phosphorylation, glycosylation); Splice variants and isoforms; Relative charge; Multimerization.

Positive Control	Mouse uterus tissue; Human colon tissue; Rat uterus tissue; Human pancreas tissue; Mouse colon tissue; Human esophagus tissue; Rat colon tissue; Human bladder cancer tissue; Human uterus tissue; Human liver tissue; Human placenta tissue; MG-63 cells
Negative Control	HT-1080 cells

Experimental Methods

WB
Experimental Protocol: Sample preparation 1. Tissue: Lyse the tissue sample by adding an appropriate volume of ice-cold RIPA/NP-40 Lysis Buffer (containing Protease Inhibitor Cocktail)，and homogenize the tissue at a low temperature or lyse it by sonication on ice, then incubate on ice for 30 minutes. 2. Adherent cell: Aspirate the culture medium and wash the cells with ice-cold PBS twice. Lyse the cells by adding an appropriate volume of RIPA/NP-40 Lysis Buffer (containing Protease Inhibitor Cocktail) , sonicate to lyse the cells, and incubate on ice for 30 minutes. 3. Suspension cell: Transfer the culture medium to a pre-cooled centrifuge tube. Centrifuge and aspirate the supernatant. Wash the cells with ice-cold PBS twice. Lyse the cells by adding an appropriate volume of RIPA/NP-40 Lysis Buffer (containing Protease Inhibitor Cocktail) , sonicate to lyse the cells, and incubate on ice for 30 minutes. 4. Place the lysate into a pre-cooled microcentrifuge tube. Centrifuge at 4°C for 15 min. Collect the supernatant; 5. Remove a small volume of lysate to determine the protein concentration; 6. Combine the lysate with protein loading buffer. Boil 20 µL sample under 95-100°C for 5 min. Centrifuge for 5 min after cool down on ice. Electrophoretic separation 1. According to the concentration of extracted protein, load appropriate amount of protein sample and marker onto SDS-PAGE gels for electrophoresis. Recommended separating gel (lower gel) concentration: 10%. Reference Table for Selecting SDS-PAGE Separation Gel Concentrations 2. Power up 80V for 30 minutes. Then the power supply is adjusted (110 V~150 V), the Marker is observed, and the electrophoresis can be stopped when the indicator band of the predyed protein Marker where the protein is located is properly separated. (Note that the current should not be too large when electrophoresis, too large current (more than 150 mA) will cause the temperature to rise, affecting the result of running glue. If high currents cannot be avoided, an ice bath can be used to cool the bath.) Transfer membrane 1. Take out the converter, soak the clip and consumables in the pre-cooled converter; 2. Activate PVDF membrane with methanol for 1 min and rinse with transfer buffer; 3. Install it in the order of "black edge of clip - sponge - filter paper - filter paper - glue -PVDF membrane - filter paper - filter paper - sponge - white edge of clip"; 4. The protein was electrotransferred to PVDF membrane. ( 0.45 µm PVDF membrane is recommended ) Reference Table for Selecting PVDF Membrane Pore Size Specifications Recommended conditions for wet transfer: 200 mA, 60 min. ( Note that the transfer conditions can be adjusted according to the protein size. For high-molecular-weight proteins, a higher current and longer transfer time are recommended. However, ensure that the transfer tank remains at a low temperature to prevent gel melting.) Block 1. After electrotransfer, wash the film with TBST at room temperature for 5 minutes; 2. Incubate the film in the blocking solution for 1 hour at room temperature; 3. Wash the film with TBST for 3 times, 5 minutes each time. Antibody incubation 1. Use 5% skim milk powder to prepare the primary antibody working liquid (recommended dilution ratio for primary antibody 1:1000), gently shake and incubate with the film at 4°C overnight; 2. Wash the film with TBST 3 times, 5 minutes each time; 3. Add the secondary antibody to the blocking solution and incubate with the film gently at room temperature for 1 hour; 4. After incubation, wash the film with TBST 3 times for 5 minutes each time. Antibody staining 1. Add the prepared ECL luminescent substrate (or select other color developing substrate according to the second antibody) and mix evenly; 2. Incubate with the film for 1 minute, remove excess substrate (keep the film moist), wrap with plastic film, and expose in the imaging system.

WB

Experimental Protocol:

Sample preparation

1. Tissue: Lyse the tissue sample by adding an appropriate volume of ice-cold RIPA/NP-40 Lysis Buffer (containing Protease Inhibitor Cocktail)，and homogenize the tissue at a low temperature or lyse it by sonication on ice, then incubate on ice for 30 minutes.

2. Adherent cell: Aspirate the culture medium and wash the cells with ice-cold PBS twice. Lyse the cells by adding an appropriate volume of RIPA/NP-40 Lysis Buffer (containing Protease Inhibitor Cocktail) , sonicate to lyse the cells, and incubate on ice for 30 minutes.

3. Suspension cell: Transfer the culture medium to a pre-cooled centrifuge tube. Centrifuge and aspirate the supernatant. Wash the cells with ice-cold PBS twice. Lyse the cells by adding an appropriate volume of RIPA/NP-40 Lysis Buffer (containing Protease Inhibitor Cocktail) , sonicate to lyse the cells, and incubate on ice for 30 minutes.

4. Place the lysate into a pre-cooled microcentrifuge tube. Centrifuge at 4°C for 15 min. Collect the supernatant;

5. Remove a small volume of lysate to determine the protein concentration;

6. Combine the lysate with protein loading buffer. Boil 20 µL sample under 95-100°C for 5 min. Centrifuge for 5 min after cool down on ice.

Electrophoretic separation

1. According to the concentration of extracted protein, load appropriate amount of protein sample and marker onto SDS-PAGE gels for electrophoresis. Recommended separating gel (lower gel) concentration: 10%. Reference Table for Selecting SDS-PAGE Separation Gel Concentrations

2. Power up 80V for 30 minutes. Then the power supply is adjusted (110 V~150 V), the Marker is observed, and the electrophoresis can be stopped when the indicator band of the predyed protein Marker where the protein is located is properly separated. (Note that the current should not be too large when electrophoresis, too large current (more than 150 mA) will cause the temperature to rise, affecting the result of running glue. If high currents cannot be avoided, an ice bath can be used to cool the bath.)

Transfer membrane

1. Take out the converter, soak the clip and consumables in the pre-cooled converter;

2. Activate PVDF membrane with methanol for 1 min and rinse with transfer buffer;

3. Install it in the order of "black edge of clip - sponge - filter paper - filter paper - glue -PVDF membrane - filter paper - filter paper - sponge - white edge of clip";

4. The protein was electrotransferred to PVDF membrane. ( 0.45 µm PVDF membrane is recommended ) Reference Table for Selecting PVDF Membrane Pore Size Specifications

Recommended conditions for wet transfer: 200 mA, 60 min.

( Note that the transfer conditions can be adjusted according to the protein size. For high-molecular-weight proteins, a higher current and longer transfer time are recommended. However, ensure that the transfer tank remains at a low temperature to prevent gel melting.)

Block

1. After electrotransfer, wash the film with TBST at room temperature for 5 minutes;

2. Incubate the film in the blocking solution for 1 hour at room temperature;

3. Wash the film with TBST for 3 times, 5 minutes each time.

Antibody incubation

1. Use 5% skim milk powder to prepare the primary antibody working liquid (recommended dilution ratio for primary antibody 1:1000), gently shake and incubate with the film at 4°C overnight;

2. Wash the film with TBST 3 times, 5 minutes each time;

3. Add the secondary antibody to the blocking solution and incubate with the film gently at room temperature for 1 hour;

4. After incubation, wash the film with TBST 3 times for 5 minutes each time.

Antibody staining

1. Add the prepared ECL luminescent substrate (or select other color developing substrate according to the second antibody) and mix evenly;

2. Incubate with the film for 1 minute, remove excess substrate (keep the film moist), wrap with plastic film, and expose in the imaging system.

IHC
Experimental Protocol： Deparaffinization/Rehydration 1. Deparaffinize/hydrate sections: 2. Incubate sections in three washes of xylene for 5 min each. 3. Incubate sections in two washes of 100% ethanol for 10 min each. 4. Incubate sections in two washes of 95% ethanol for 10 min each. 5. Wash sections two times in dH2O for 5 min each. 6.Antigen retrieval: For Citrate: Heat slides in a microwave submersed in 1X citrate unmasking solution until boiling is initiated; continue with 10 min at a sub-boiling temperature (95°-98°C). Cool slides on bench top for 30 min. Staining 1. Wash sections in dH2O three times for 5 min each. 2. Incubate sections in 3% hydrogen peroxide for 10 min. 3. Wash sections in dH2O two times for 5 min each. 4. Wash sections in wash buffer for 5 min. 5. Block each section with 100–400 µl of blocking solution for 1 hr at room temperature. 6. Remove blocking solution and add 100–400 µl primary antibody diluent in to each section. Incubate overnight at 4°C. 7. Remove antibody solution and wash sections with wash buffer three times for 5 min each. 8. Cover section with 1–3 drops HRPas needed. Incubate in a humidified chamber for 30 min at room temperature. 9. Wash sections three times with wash buffer for 5 min each. 10. Add DAB Chromogen Concentrate to DAB Diluent and mix well before use. 11. Apply 100–400 µl DAB to each section and monitor closely. 1–10 min generally provides an acceptable staining intensity. 12. Immerse slides in dH2O. 13. If desired, counterstain sections with hematoxylin. 14. Wash sections in dH2O two times for 5 min each. 15. Dehydrate sections: Incubate sections in 95% ethanol two times for 10 sec each; Repeat in 100% ethanol, incubating sections two times for 10 sec each; Repeat in xylene, incubating sections two times for 10 sec each. 16. Mount sections with coverslips and mounting medium.

IHC

Experimental Protocol：

Deparaffinization/Rehydration

1. Deparaffinize/hydrate sections:

2. Incubate sections in three washes of xylene for 5 min each.

3. Incubate sections in two washes of 100% ethanol for 10 min each.

4. Incubate sections in two washes of 95% ethanol for 10 min each.

5. Wash sections two times in dH2O for 5 min each.

6.Antigen retrieval: For Citrate: Heat slides in a microwave submersed in 1X citrate unmasking solution until boiling is initiated; continue with 10 min at a sub-boiling temperature (95°-98°C). Cool slides on bench top for 30 min.

Staining

1. Wash sections in dH2O three times for 5 min each.

2. Incubate sections in 3% hydrogen peroxide for 10 min.

3. Wash sections in dH2O two times for 5 min each.

4. Wash sections in wash buffer for 5 min.

5. Block each section with 100–400 µl of blocking solution for 1 hr at room temperature.

6. Remove blocking solution and add 100–400 µl primary antibody diluent in to each section. Incubate overnight at 4°C.

7. Remove antibody solution and wash sections with wash buffer three times for 5 min each.

8. Cover section with 1–3 drops HRPas needed. Incubate in a humidified chamber for 30 min at room temperature.

9. Wash sections three times with wash buffer for 5 min each.

10. Add DAB Chromogen Concentrate to DAB Diluent and mix well before use.

11. Apply 100–400 µl DAB to each section and monitor closely. 1–10 min generally provides an acceptable staining intensity.

12. Immerse slides in dH2O.

13. If desired, counterstain sections with hematoxylin.

14. Wash sections in dH2O two times for 5 min each.

15. Dehydrate sections: Incubate sections in 95% ethanol two times for 10 sec each; Repeat in 100% ethanol, incubating sections two times for 10 sec each; Repeat in xylene, incubating sections two times for 10 sec each.

16. Mount sections with coverslips and mounting medium.

Datasheet & SDS

Biological Description

Specificity
Decorin Antibody (Rabbit mAb) [E13A13] detects endogenous levels of total Decorin protein.

Subcellular Location
Extracellular matrix, Secreted

Uniprot ID
P07585

Clone
E13A13

Synonym(s)
SLRR1B, DCN, Decorin, Bone proteoglycan II, PG-S2, PG40

Background
Decorin is a stromal-derived, small leucine-rich proteoglycan that decorates collagen fibrils in the extracellular matrix and serves as a structural and signaling hub controlling fibrillogenesis, receptor activity and tissue homeostasis. The protein core adopts a curved solenoid formed by tandem leucine-rich repeats, with an N‑terminal glycosaminoglycan attachment site and a dimerization interface that positions its concave face to bind fibrillar collagens and fibrin, and its convex surface to engage growth factors and receptor ectodomains. Interaction with collagen I and other fibrillar collagens modulates lateral assembly and spacing of fibrils, altering fibril diameter, organization and mechanical properties in tissues such as tendon, skin and cartilage, and thereby impacts matrix stiffness and load-bearing capacity. Direct binding to the D regions of fibrinogen extends this structural role to clot formation; decorin incorporation into fibrin networks reduces fiber diameter, produces thinner, more curved fibrin fibers and accelerates tissue-type plasminogen activator–dependent fibrinolysis, indicating that decorin influences both fibrin assembly and clearance during hemostasis and wound repair. As a soluble matrix component, decorin functions as a pan–receptor tyrosine kinase inhibitor by binding to multiple RTKs including EGFR, HER2, Met, VEGFR2, IGF-IR and certain Toll-like receptors, evoking transient receptor activation followed by suppression of downstream cascades such as ERK, PI3K/AKT and β‑catenin and driving receptor internalization and lysosomal degradation. Engagement of Met leads to reduced β‑catenin signaling and growth inhibition, while interaction with VEGFR2 restrains angiogenic signaling and triggers sustained endothelial autophagy, linking decorin to both tumor oncosuppression and anti-angiogenic mechanisms. In cancer models, decorin expression in the tumor stroma or exogenous decorin administration decreases tumor growth, limits invasion and curbs neovascularization through coordinated inhibition of RTKs and modulation of TGF‑β activity, establishing decorin as a potent extracellular oncosuppressor. Decorin also regulates inflammatory and immune pathways by controlling PDCD4 and related mediators and by shaping cytokine and chemokine expression, connecting its matrix-based signaling to immune surveillance and tumor–inflammation crosstalk. In cartilage, decorin stabilizes the aggrecan network by increasing adhesion between aggrecan molecules and between aggrecan and collagen II fibrils, promoting retention of aggrecan in newly synthesized matrix and supporting the compressive modulus, hydraulic permeability and energy dissipation properties required for normal joint biomechanics. Decorin’s leucine-rich repeat architecture, collagen and fibrin binding, and broad receptor interaction repertoire underpin its dual roles as a structural regulator of fibril formation and a soluble modulator of growth factor and RTK signaling, making it a mechanistically rich target for studying extracellular matrix biology, tissue repair and matrix-driven control of tumor progression.

Background

Decorin is a stromal-derived, small leucine-rich proteoglycan that decorates collagen fibrils in the extracellular matrix and serves as a structural and signaling hub controlling fibrillogenesis, receptor activity and tissue homeostasis. The protein core adopts a curved solenoid formed by tandem leucine-rich repeats, with an N‑terminal glycosaminoglycan attachment site and a dimerization interface that positions its concave face to bind fibrillar collagens and fibrin, and its convex surface to engage growth factors and receptor ectodomains. Interaction with collagen I and other fibrillar collagens modulates lateral assembly and spacing of fibrils, altering fibril diameter, organization and mechanical properties in tissues such as tendon, skin and cartilage, and thereby impacts matrix stiffness and load-bearing capacity. Direct binding to the D regions of fibrinogen extends this structural role to clot formation; decorin incorporation into fibrin networks reduces fiber diameter, produces thinner, more curved fibrin fibers and accelerates tissue-type plasminogen activator–dependent fibrinolysis, indicating that decorin influences both fibrin assembly and clearance during hemostasis and wound repair. As a soluble matrix component, decorin functions as a pan–receptor tyrosine kinase inhibitor by binding to multiple RTKs including EGFR, HER2, Met, VEGFR2, IGF-IR and certain Toll-like receptors, evoking transient receptor activation followed by suppression of downstream cascades such as ERK, PI3K/AKT and β‑catenin and driving receptor internalization and lysosomal degradation. Engagement of Met leads to reduced β‑catenin signaling and growth inhibition, while interaction with VEGFR2 restrains angiogenic signaling and triggers sustained endothelial autophagy, linking decorin to both tumor oncosuppression and anti-angiogenic mechanisms. In cancer models, decorin expression in the tumor stroma or exogenous decorin administration decreases tumor growth, limits invasion and curbs neovascularization through coordinated inhibition of RTKs and modulation of TGF‑β activity, establishing decorin as a potent extracellular oncosuppressor. Decorin also regulates inflammatory and immune pathways by controlling PDCD4 and related mediators and by shaping cytokine and chemokine expression, connecting its matrix-based signaling to immune surveillance and tumor–inflammation crosstalk. In cartilage, decorin stabilizes the aggrecan network by increasing adhesion between aggrecan molecules and between aggrecan and collagen II fibrils, promoting retention of aggrecan in newly synthesized matrix and supporting the compressive modulus, hydraulic permeability and energy dissipation properties required for normal joint biomechanics. Decorin’s leucine-rich repeat architecture, collagen and fibrin binding, and broad receptor interaction repertoire underpin its dual roles as a structural regulator of fibril formation and a soluble modulator of growth factor and RTK signaling, making it a mechanistically rich target for studying extracellular matrix biology, tissue repair and matrix-driven control of tumor progression.

References
https://pubmed.ncbi.nlm.nih.gov/22735579/ https://pubmed.ncbi.nlm.nih.gov/17046817/

Protein Size (kDa)	PVDF Transfer Membrane Pore Size (μm)
< 10	0.22
10-20	0.22
20-30	0.45
30-45	0.45
45-70	0.45
80-100	0.45
100-140	0.45
> 140	0.45

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Protein Size (kDa)	Separating Gel Percentage
4-40	20%
12-45	15%
10-70	12.5%
15-100	10%
25-100	8%
60-210	5%