Aggrecan Antibody [M12J8] | Primary Antibodies

Application: Reactivity: Human

Lane 1: A375

Experiment Essentials

WB
Recommended SDS-PAGE separating gel concentration: 5%.
Recommended wet transfer conditions: 250 mA, 180 min.
Exposure time of at least 60s is recommended.

Usage Information

Dilution
WB1:1000-1:10000

Application
WB

Reactivity
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
261 kDa 600 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	A-375 cells (PNGaseF treated); Hs 822.T cells (PNGaseF treated); Hs 822.T cells; A-375 cells
Negative Control	Hep G2 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: 5%. 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: 250 mA, 180 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. (Exposure time of at least 60s is recommended)

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: 5%. 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: 250 mA, 180 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. (Exposure time of at least 60s is recommended)

Datasheet & SDS

Biological Description

Specificity
Aggrecan Antibody (Rabbit mAb) [M12J8] detects endogenous levels of total Aggrecan protein.

Subcellular Location
Extracellular matrix, Secreted

Uniprot ID
P16112

Clone
M12J8

Synonym(s)
ACAN, AGC1, AGCAN, aggrecan, Aggrecan core protein, Aggrecan core protein 2, CSPCP, CSPG1, CSPGCP, large aggregating proteoglycan, MSK16, PGCA, SEDK, SSOAOD

Background
Aggrecan, encoded by ACAN, is a large chondroitin sulfate proteoglycan of the lectican family that is highly expressed in growth plate and articular cartilage and forms a major non‑collagenous structural component of the cartilage extracellular matrix together with type II collagen. The core protein contains three globular domains (G1, G2, G3) separated by extended regions bearing dense arrays of chondroitin sulfate and keratan sulfate chains; the N‑terminal G1 domain, which shares structural motifs with link protein, binds hyaluronan and link protein to form large ternary aggregates, the central G2 domain is homologous to G1/link repeats and contributes to product processing, and the C‑terminal G3 domain regulates glycosaminoglycan modification, secretion, and interactions with other matrix molecules. The glycosaminoglycan‑rich domain between G2 and G3 carries multiple chondroitin sulfate and keratan sulfate chains that create a highly negatively charged, bottlebrush‑like polyelectrolyte, and aggrecan–hyaluronan aggregates embedded in the collagen network form a hydrated gel that imbibes water, generates swelling pressure, and provides the osmotic and mechanical properties required for cartilage to resist compressive loads during joint use. Through hyaluronan‑dependent aggregation and interactions of its G3 domain with ECM partners such as tenascins, fibulins, and fibrillin, aggrecan organizes the pericellular and territorial matrix and contributes to chondrocyte–matrix and chondrocyte–chondrocyte interactions that influence chondroskeletal morphogenesis in development and maintenance of articular cartilage architecture in maturity. In the central nervous system, aggrecan is present in perineuronal nets where it binds hyaluronan, link protein, and tenascins to form condensed ECM structures around neurons, and in this context it regulates the organization of neural extracellular space and contributes to control of developmental plasticity and responses to injury. ACAN mutations that alter core protein structure or glycosaminoglycan attachment associate with short stature, skeletal dysplasia, and abnormal joint and spine development, consistent with a central role of aggrecan in growth plate function and cartilage patterning. In osteoarthritis and related degenerative joint diseases, aggrecan loss from cartilage is an early and prominent event, and cleavage of the core protein by aggrecanases such as ADAMTS‑4 and ADAMTS‑5, and by matrix metalloproteinases, generates defined fragments that diffuse from the tissue and reduce proteoglycan content, leading to diminished load‑bearing capacity and altered cartilage biomechanics.

Background

Aggrecan, encoded by ACAN, is a large chondroitin sulfate proteoglycan of the lectican family that is highly expressed in growth plate and articular cartilage and forms a major non‑collagenous structural component of the cartilage extracellular matrix together with type II collagen. The core protein contains three globular domains (G1, G2, G3) separated by extended regions bearing dense arrays of chondroitin sulfate and keratan sulfate chains; the N‑terminal G1 domain, which shares structural motifs with link protein, binds hyaluronan and link protein to form large ternary aggregates, the central G2 domain is homologous to G1/link repeats and contributes to product processing, and the C‑terminal G3 domain regulates glycosaminoglycan modification, secretion, and interactions with other matrix molecules. The glycosaminoglycan‑rich domain between G2 and G3 carries multiple chondroitin sulfate and keratan sulfate chains that create a highly negatively charged, bottlebrush‑like polyelectrolyte, and aggrecan–hyaluronan aggregates embedded in the collagen network form a hydrated gel that imbibes water, generates swelling pressure, and provides the osmotic and mechanical properties required for cartilage to resist compressive loads during joint use. Through hyaluronan‑dependent aggregation and interactions of its G3 domain with ECM partners such as tenascins, fibulins, and fibrillin, aggrecan organizes the pericellular and territorial matrix and contributes to chondrocyte–matrix and chondrocyte–chondrocyte interactions that influence chondroskeletal morphogenesis in development and maintenance of articular cartilage architecture in maturity. In the central nervous system, aggrecan is present in perineuronal nets where it binds hyaluronan, link protein, and tenascins to form condensed ECM structures around neurons, and in this context it regulates the organization of neural extracellular space and contributes to control of developmental plasticity and responses to injury. ACAN mutations that alter core protein structure or glycosaminoglycan attachment associate with short stature, skeletal dysplasia, and abnormal joint and spine development, consistent with a central role of aggrecan in growth plate function and cartilage patterning. In osteoarthritis and related degenerative joint diseases, aggrecan loss from cartilage is an early and prominent event, and cleavage of the core protein by aggrecanases such as ADAMTS‑4 and ADAMTS‑5, and by matrix metalloproteinases, generates defined fragments that diffuse from the tissue and reduce proteoglycan content, leading to diminished load‑bearing capacity and altered cartilage biomechanics.

References
https://pubmed.ncbi.nlm.nih.gov/11942407/ https://pubmed.ncbi.nlm.nih.gov/26914753/

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%

Aggrecan Antibody (Rabbit mAb) [M12J8]