Rubella Virus capsid Antibody [M17H6]

Application: Reactivity: Rubella virus

Lane 1: recombinant purified rubella capsid protein, Lane 2: irrelevant viral proteins

Experiment Essentials

WB
Recommended SDS-PAGE separating gel concentration: 5%.

Usage Information

Dilution
WB1:1000 - 1:5000

Application
WB, ELISA

Reactivity
Rubella virus

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
115 kDa

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 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: 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 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.

Datasheet & SDS

Biological Description

Specificity
Rubella Virus capsid Antibody (Mouse mAb) [M17H6] detects exogenous levels of total Rubella Virus capsid protein.

Subcellular Location
Capsid protein, Host cytoplasm, Host Golgi apparatus, Host membrane, Host mitochondrion, T=4 icosahedral capsid protein

Uniprot ID
P07566

Clone
M17H6

Synonym(s)
Structural polyprotein, p110, Structural polyprotein, p110

Background
Rubella virus capsid is the sole capsid protein of the rubella virus within the Rubivirus genus and functions as the core structural component that packages the positive-strand RNA genome and organizes virion assembly at intracellular membranes. The protein contains an N-terminal structural domain that forms the capsid protomer with a unique fold not observed in other viral capsids, and a more flexible C-terminal segment that carries basic and membrane-proximal elements important for RNA binding and interaction with the viral envelope glycoproteins. Capsid binds genomic RNA to form ribonucleoprotein cores and associates with the E1–E2 glycoprotein complex at Golgi-derived membranes, where coordinated interactions between its structural domain and the cytoplasmic tails of glycoproteins drive budding and generation of enveloped virions with defined core geometry. The three-dimensional arrangement of capsid protomers within the virion supports formation of an icosahedrally ordered core that stabilizes the RNA, positions it relative to the envelope, and contributes to overall particle integrity during extracellular transmission. Capsid also participates in virus–host communication by engaging host factors that influence translation and innate defense pathways, including interactions with cellular poly(A)-binding protein that modulate host translation and favor viral gene expression. Phosphorylation of defined residues in the capsid regulates RNA-binding capacity and nucleocapsid assembly, adding a layer of post-translational control that links the capsid–RNA interaction to the timing and efficiency of virion formation. During the rubella virus life cycle, the capsid functions together with nonstructural replicase components and envelope proteins to coordinate replication complex activity, nucleocapsid formation, and budding, embedding it at the interface between genome replication and particle morphogenesis. In infected tissues, the capsid contributes to persistence of viral RNA and maintenance of infectious virions, a property that is relevant to the teratogenic potential of the rubella virus and the pathogenesis of congenital rubella syndrome when infection occurs during early pregnancy.

Background

Rubella virus capsid is the sole capsid protein of the rubella virus within the Rubivirus genus and functions as the core structural component that packages the positive-strand RNA genome and organizes virion assembly at intracellular membranes. The protein contains an N-terminal structural domain that forms the capsid protomer with a unique fold not observed in other viral capsids, and a more flexible C-terminal segment that carries basic and membrane-proximal elements important for RNA binding and interaction with the viral envelope glycoproteins. Capsid binds genomic RNA to form ribonucleoprotein cores and associates with the E1–E2 glycoprotein complex at Golgi-derived membranes, where coordinated interactions between its structural domain and the cytoplasmic tails of glycoproteins drive budding and generation of enveloped virions with defined core geometry. The three-dimensional arrangement of capsid protomers within the virion supports formation of an icosahedrally ordered core that stabilizes the RNA, positions it relative to the envelope, and contributes to overall particle integrity during extracellular transmission. Capsid also participates in virus–host communication by engaging host factors that influence translation and innate defense pathways, including interactions with cellular poly(A)-binding protein that modulate host translation and favor viral gene expression. Phosphorylation of defined residues in the capsid regulates RNA-binding capacity and nucleocapsid assembly, adding a layer of post-translational control that links the capsid–RNA interaction to the timing and efficiency of virion formation. During the rubella virus life cycle, the capsid functions together with nonstructural replicase components and envelope proteins to coordinate replication complex activity, nucleocapsid formation, and budding, embedding it at the interface between genome replication and particle morphogenesis. In infected tissues, the capsid contributes to persistence of viral RNA and maintenance of infectious virions, a property that is relevant to the teratogenic potential of the rubella virus and the pathogenesis of congenital rubella syndrome when infection occurs during early pregnancy.

References
https://pubmed.ncbi.nlm.nih.gov/24282305/ https://pubmed.ncbi.nlm.nih.gov/33627388/

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%

Rubella Virus capsid Antibody (Mouse mAb) [M17H6]