Mouse IgG Antibody [B6P15] | Primary Antibodies

Application: Reactivity: Mouse

Lane 1: Recombinant IgG1 Protein

Experiment Essentials

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

Usage Information

Dilution
WB1:2000-1:10000

Application
WB, ELISA

Reactivity
Mouse

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
38 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. 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) and put the sample on ice for 5 min. 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) and put the sample on ice for 5 min. 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 1:2000), 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.

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) and put the sample on ice for 5 min.

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) and put the sample on ice for 5 min.

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 1:2000), 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.

Datasheet & SDS

Biological Description

Specificity
Mouse IgG Antibody [B6P15] detects endogenous levels of total IgG protein.

Subcellular Location
Cell membrane, Immunoglobulin, Membrane, Secreted

Uniprot ID
P01857

Clone
B6P15

Synonym(s)
Constant region of heavy chain of IgG1; G1m marker; Gm marker; Ig gamma 1 chain C region; Immunoglobin heavy constant gamma 1; Immunoglobulin gamma 1

Background
Mouse IgG is the predominant immunoglobulin isotype in mouse serum, with four subclasses, IgG1, IgG2a, IgG2b, and IgG3, produced by plasma B cells as part of the humoral immune response. Each IgG molecule consists of two identical γ heavy chains and two light chains (κ or λ), forming a characteristic Y-shaped structure with antigen-binding Fab regions linked via a flexible, proline- and cysteine-rich hinge to the Fc region (CH2-CH3 domains with conserved N-glycosylation). Subclass differences in hinge length and disulfide bonding influence molecular flexibility and affinity for Fcγ receptors (FcγRI–III), which in turn dictate effector functions. Mouse IgG neutralizes pathogens by antigen binding, opsonizes targets via complement activation (notably IgG2a and IgG2b), and mediates antibody-dependent cellular cytotoxicity (ADCC) through distinct FcγR interactions: IgG2a is highly effective at both complement activation and ADCC, IgG1 is associated with Th2 responses and modest effector activity, IgG3 provides strong opsonization, and IgG2b balances phagocytosis and cytotoxicity. These functions drive cytokine production (such as IFNγ and IL-4), promote immune complex clearance, establish B cell memory, and regulate innate immune responses through neonatal FcRn-mediated recycling that extends serum half-life. Subclass imbalances are implicated in autoimmune diseases (e.g., IgG2a in lupus models), allergies (IgG1), and antitumor immunity, while conserved Fc glycosylation patterns further modulate inflammatory potential.

Background

Mouse IgG is the predominant immunoglobulin isotype in mouse serum, with four subclasses, IgG1, IgG2a, IgG2b, and IgG3, produced by plasma B cells as part of the humoral immune response. Each IgG molecule consists of two identical γ heavy chains and two light chains (κ or λ), forming a characteristic Y-shaped structure with antigen-binding Fab regions linked via a flexible, proline- and cysteine-rich hinge to the Fc region (CH2-CH3 domains with conserved N-glycosylation). Subclass differences in hinge length and disulfide bonding influence molecular flexibility and affinity for Fcγ receptors (FcγRI–III), which in turn dictate effector functions. Mouse IgG neutralizes pathogens by antigen binding, opsonizes targets via complement activation (notably IgG2a and IgG2b), and mediates antibody-dependent cellular cytotoxicity (ADCC) through distinct FcγR interactions: IgG2a is highly effective at both complement activation and ADCC, IgG1 is associated with Th2 responses and modest effector activity, IgG3 provides strong opsonization, and IgG2b balances phagocytosis and cytotoxicity. These functions drive cytokine production (such as IFNγ and IL-4), promote immune complex clearance, establish B cell memory, and regulate innate immune responses through neonatal FcRn-mediated recycling that extends serum half-life. Subclass imbalances are implicated in autoimmune diseases (e.g., IgG2a in lupus models), allergies (IgG1), and antitumor immunity, while conserved Fc glycosylation patterns further modulate inflammatory potential.

References
https://pubmed.ncbi.nlm.nih.gov/25368619/ https://pubmed.ncbi.nlm.nih.gov/18064051/

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%