MUC2 Antibody (Rabbit mAb) [M5E4] Datasheet

Biological Description

Specificity	MUC2 Antibody (Rabbit mAb) [M5E4] detects endogenous levels of total MUC2 protein.
Background	MUC2 belongs to the gel-forming mucin family, related structurally to von Willebrand factor, and serves as the principal building block of the intestinal mucus layers that physically separate the gut microbiota from the underlying epithelial surface. The protein carries an N-terminal region built from tandem von Willebrand D domains, VWD1, VWD2, and VWD'D3, interspersed with cysteine-rich CysD domains, a central region dominated by heavily O-glycosylated PTS domains rich in serine, threonine, and proline, and a C-terminal region containing a further VWD4 domain followed by roughly three and a half linear VWC domains and a terminal cystine-knot domain. Following synthesis, MUC2 first dimerizes in the endoplasmic reticulum through disulfide bonds formed between the cystine-knot domains at the C-termini of two monomers, and this C-terminal dimer is then translocated to the Golgi apparatus, where extensive O-glycosylation of the central PTS domains expands the molecule to a mass in the megadalton range. A structural model of the MUC2 C-terminal dimer, built by combining cryo-electron microscopy, computational structure prediction, and small-angle X-ray scattering, reveals that all gel-forming mucins and von Willebrand factor share the same tail-to-tail disulfide-linked dimer architecture at the cystine-knot domain, but MUC2 specifically carries an additional stabilizing disulfide bond positioned on the N-terminal side of its VWD4 domain, a feature not present in the other gel-forming mucins or in von Willebrand factor and proposed to be specifically required for the stability of the intestinal mucus barrier. Once C-terminal dimers are formed, a separate assembly step links the N-termini of multiple dimers together through additional disulfide bonds, producing a covalently connected polymeric network that is packaged into mucin granules within goblet cells before secretion. After secretion, this disulfide-stabilized polymeric skeleton undergoes proteolytic cleavage of the MUC2 protein core, and because these cleavage events occur without disrupting the underlying disulfide-bonded polymeric network, the mucus can expand substantially in volume while its structural continuity is preserved, converting the compact, firmly adherent mucus layer into the more voluminous, loosely organized outer layer. The resulting two-layer colonic mucus system depends entirely on this MUC2 polymer network: the inner layer remains firmly attached, compact, and impermeable to bacteria, physically excluding the intestinal microbiota from direct contact with the epithelium, while the outer layer, generated through proteolytic expansion of the same MUC2 network, provides the habitat in which commensal bacteria reside, and this microbial exclusion function of the inner MUC2 layer occurs independently of adaptive or innate immune mechanisms, since it is preserved even in germ-free animals. Disruption of MUC2 network assembly, folding, or its bacteria-excluding barrier function is implicated in ulcerative colitis, positioning both the disulfide-mediated polymerization interfaces and the proteolytically expandable network architecture as defined structural features relevant to intestinal barrier disease.

Specificity

MUC2 Antibody (Rabbit mAb) [M5E4] detects endogenous levels of total MUC2 protein.

Background

MUC2 belongs to the gel-forming mucin family, related structurally to von Willebrand factor, and serves as the principal building block of the intestinal mucus layers that physically separate the gut microbiota from the underlying epithelial surface. The protein carries an N-terminal region built from tandem von Willebrand D domains, VWD1, VWD2, and VWD'D3, interspersed with cysteine-rich CysD domains, a central region dominated by heavily O-glycosylated PTS domains rich in serine, threonine, and proline, and a C-terminal region containing a further VWD4 domain followed by roughly three and a half linear VWC domains and a terminal cystine-knot domain. Following synthesis, MUC2 first dimerizes in the endoplasmic reticulum through disulfide bonds formed between the cystine-knot domains at the C-termini of two monomers, and this C-terminal dimer is then translocated to the Golgi apparatus, where extensive O-glycosylation of the central PTS domains expands the molecule to a mass in the megadalton range. A structural model of the MUC2 C-terminal dimer, built by combining cryo-electron microscopy, computational structure prediction, and small-angle X-ray scattering, reveals that all gel-forming mucins and von Willebrand factor share the same tail-to-tail disulfide-linked dimer architecture at the cystine-knot domain, but MUC2 specifically carries an additional stabilizing disulfide bond positioned on the N-terminal side of its VWD4 domain, a feature not present in the other gel-forming mucins or in von Willebrand factor and proposed to be specifically required for the stability of the intestinal mucus barrier. Once C-terminal dimers are formed, a separate assembly step links the N-termini of multiple dimers together through additional disulfide bonds, producing a covalently connected polymeric network that is packaged into mucin granules within goblet cells before secretion. After secretion, this disulfide-stabilized polymeric skeleton undergoes proteolytic cleavage of the MUC2 protein core, and because these cleavage events occur without disrupting the underlying disulfide-bonded polymeric network, the mucus can expand substantially in volume while its structural continuity is preserved, converting the compact, firmly adherent mucus layer into the more voluminous, loosely organized outer layer. The resulting two-layer colonic mucus system depends entirely on this MUC2 polymer network: the inner layer remains firmly attached, compact, and impermeable to bacteria, physically excluding the intestinal microbiota from direct contact with the epithelium, while the outer layer, generated through proteolytic expansion of the same MUC2 network, provides the habitat in which commensal bacteria reside, and this microbial exclusion function of the inner MUC2 layer occurs independently of adaptive or innate immune mechanisms, since it is preserved even in germ-free animals. Disruption of MUC2 network assembly, folding, or its bacteria-excluding barrier function is implicated in ulcerative colitis, positioning both the disulfide-mediated polymerization interfaces and the proteolytically expandable network architecture as defined structural features relevant to intestinal barrier disease.

Usage Information

Application

WB, IP, ELISA

Dilution

WB	IP
1:500-1:1000	1:15000

Reactivity

Human

Source

Rabbit Monoclonal Antibody

MW

551 kDa

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

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: 200 mA, 120 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 1:1000), 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.

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: 200 mA, 120 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 1:1000), 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.

References

https://pubmed.ncbi.nlm.nih.gov/20615996/
https://pubmed.ncbi.nlm.nih.gov/37031240/

Application Data

WB

Validated by Selleck

Lane 1: Caco-2