SDC2 Antibody [D5J9] | Primary Antibodies

Application: Reactivity: Human

Lane 1: HuH7, Lane 2: HepG2, Lane 3: A549, Lane 4: Jurkat

Experiment Essentials

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

Usage Information

Dilution
WB1:2000-1:10000 IHC1:250-1:1000

Application
WB, IHC

Reactivity
Human

Source
Mouse 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
22 kDa 19 kDa, 48 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	Human liver cancer tissue; L02 cells; Huh‑7 cells; HepG2 cells; A549 cells; NCI‑H1299 cells; Jurkat cells
Negative Control

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 primary antibody dilution buffer to prepare the primary antibody working liquid (recommended dilution ratio for primary antibody 1:2000), 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 primary antibody dilution buffer to prepare the primary antibody working liquid (recommended dilution ratio for primary antibody 1:2000), 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
SDC2 Antibody (Mouse mAb) [D5J9] detects endogenous levels of total SDC2 protein.

Subcellular Location
Membrane

Uniprot ID
P34741

Clone
D5J9

Synonym(s)
CD362 \| HSPG1 \| SDC2 \| Syndecan-2 \| SYND2 \| Fibroglycan \| Heparan sulfate proteoglycan core protein \| HSPG

Background
SDC2 (syndecan‑2) is a type I transmembrane heparan sulfate proteoglycan of the syndecan family that decorates its extracellular core protein with heparan sulfate chains and functions as a multifunctional cell-surface co-receptor integrating extracellular matrix, growth factor, and morphogen signals with cytoskeletal organization and intracellular kinase pathways. The protein comprises a short N‑terminal signal peptide, a luminal extracellular domain bearing heparan sulfate attachment sites, a single transmembrane segment, and a cytoplasmic tail with conserved C1 and C2 motifs and a variable region that binds PDZ-domain scaffolds and signaling adaptors; this modular structure allows SDC2 to cluster in membrane microdomains and assemble signaling complexes at sites of cell–matrix and cell–cell contact. Heparan sulfate chains on SDC2 bind a range of ligands including fibronectin, thrombospondin, FGF2, EGF-family ligands, and other ECM and growth factors, and these interactions facilitate ligand presentation to receptor tyrosine kinases and integrins, modulate ligand gradients, and promote integrin activation, PI3K–Akt and Ras–Raf–MEK–ERK signaling, and reorganization of the actin cytoskeleton that together support cell proliferation, adhesion, and directional migration. In neuronal tissue, SDC2 is enriched postsynaptically and regulates dendritic spine and arbor morphogenesis; postsynaptic SDC2 recruits and organizes FGF22-dependent transsynaptic signaling that coordinates presynaptic and postsynaptic assembly, illustrating how SDC2 functions as a synaptic organizer that couples heparan sulfate–mediated ligand binding with local cytoskeletal and receptor rearrangements. During development and epithelial morphogenesis, SDC2 participates in FGF2- and FGF receptor–dependent signaling in specialized epithelia, and in multiple systems it influences branching morphogenesis, angiogenesis, and tissue patterning by regulating local availability and receptor engagement of growth factors and by modulating downstream ERK and PI3K pathways. In colorectal cancer, SDC2 expression is significantly upregulated and the protein promotes proliferation, migration, invasion, and epithelial–mesenchymal transition, with SDC2 knockdown reducing p‑MEK/MEK and p‑ERK/ERK ratios and reversing EMT marker changes, which indicates that SDC2 amplifies MAPK signaling and EMT programs to support tumor progression. Additional work shows that SDC2 contributes to 5‑fluorouracil resistance in colorectal cancer by acting downstream of miR‑20b‑5p and engaging the JNK/ERK signaling pathway; SDC2 overexpression increases survival of 5‑FU–treated cells, whereas SDC2 suppression restores drug sensitivity, defining a miR‑20b‑5p/SDC2–JNK/ERK axis that links SDC2 expression to chemoresistance. SDC2 is also enriched in cancer-derived extracellular vesicles and co‑packages with fibronectin and other cargo, supporting roles in vesicle biogenesis and intercellular communication that propagate pro-metastatic signals in the tumor microenvironment. SDC2 expression in the nervous system and other tissues ties it to dendritic and synaptic development, wound healing, angiogenesis, and immune signaling, and SDC2 gene methylation is being exploited as a noninvasive epigenetic biomarker for early detection of colorectal neoplasia, where hypermethylated SDC2 DNA in stool or plasma reflects tumor-associated epigenetic alteration of this signaling proteoglycan.

Background

SDC2 (syndecan‑2) is a type I transmembrane heparan sulfate proteoglycan of the syndecan family that decorates its extracellular core protein with heparan sulfate chains and functions as a multifunctional cell-surface co-receptor integrating extracellular matrix, growth factor, and morphogen signals with cytoskeletal organization and intracellular kinase pathways. The protein comprises a short N‑terminal signal peptide, a luminal extracellular domain bearing heparan sulfate attachment sites, a single transmembrane segment, and a cytoplasmic tail with conserved C1 and C2 motifs and a variable region that binds PDZ-domain scaffolds and signaling adaptors; this modular structure allows SDC2 to cluster in membrane microdomains and assemble signaling complexes at sites of cell–matrix and cell–cell contact. Heparan sulfate chains on SDC2 bind a range of ligands including fibronectin, thrombospondin, FGF2, EGF-family ligands, and other ECM and growth factors, and these interactions facilitate ligand presentation to receptor tyrosine kinases and integrins, modulate ligand gradients, and promote integrin activation, PI3K–Akt and Ras–Raf–MEK–ERK signaling, and reorganization of the actin cytoskeleton that together support cell proliferation, adhesion, and directional migration. In neuronal tissue, SDC2 is enriched postsynaptically and regulates dendritic spine and arbor morphogenesis; postsynaptic SDC2 recruits and organizes FGF22-dependent transsynaptic signaling that coordinates presynaptic and postsynaptic assembly, illustrating how SDC2 functions as a synaptic organizer that couples heparan sulfate–mediated ligand binding with local cytoskeletal and receptor rearrangements. During development and epithelial morphogenesis, SDC2 participates in FGF2- and FGF receptor–dependent signaling in specialized epithelia, and in multiple systems it influences branching morphogenesis, angiogenesis, and tissue patterning by regulating local availability and receptor engagement of growth factors and by modulating downstream ERK and PI3K pathways. In colorectal cancer, SDC2 expression is significantly upregulated and the protein promotes proliferation, migration, invasion, and epithelial–mesenchymal transition, with SDC2 knockdown reducing p‑MEK/MEK and p‑ERK/ERK ratios and reversing EMT marker changes, which indicates that SDC2 amplifies MAPK signaling and EMT programs to support tumor progression. Additional work shows that SDC2 contributes to 5‑fluorouracil resistance in colorectal cancer by acting downstream of miR‑20b‑5p and engaging the JNK/ERK signaling pathway; SDC2 overexpression increases survival of 5‑FU–treated cells, whereas SDC2 suppression restores drug sensitivity, defining a miR‑20b‑5p/SDC2–JNK/ERK axis that links SDC2 expression to chemoresistance. SDC2 is also enriched in cancer-derived extracellular vesicles and co‑packages with fibronectin and other cargo, supporting roles in vesicle biogenesis and intercellular communication that propagate pro-metastatic signals in the tumor microenvironment. SDC2 expression in the nervous system and other tissues ties it to dendritic and synaptic development, wound healing, angiogenesis, and immune signaling, and SDC2 gene methylation is being exploited as a noninvasive epigenetic biomarker for early detection of colorectal neoplasia, where hypermethylated SDC2 DNA in stool or plasma reflects tumor-associated epigenetic alteration of this signaling proteoglycan.

References
https://pubmed.ncbi.nlm.nih.gov/31707342/ https://pubmed.ncbi.nlm.nih.gov/27627962/

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%

SDC2 Antibody (Mouse mAb) [D5J9]