IL-18 Antibody [K7E7] | Primary Antibodies

Application: Reactivity: Mouse

Lane 1: Mouse spleen

Experiment Essentials

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

Usage Information

Dilution
WB1:1000 IP1:40

Application
WB, IP

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
22 kDa

Positive Control	Mouse IL-18 active protein, Mouse spleen lysate
Negative Control

Exprimental 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 1:1000), 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.

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 1:1000), 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
IL-18 Antibody [K7E7] recognizes endogenous levels of total IL-18 protein.

Subcellular Location
Cytoplasm, Secreted

Uniprot ID
P70380

Clone
K7E7

Synonym(s)
Igif, Il18, Interleukin-18, IL-18, Interferon gamma-inducing factor, Interleukin-1 gamma, IFN-gamma-inducing factor, IL-1 gamma

Background
Interleukin-18 (IL-18) is a member of the interleukin-1 (IL-1) cytokine family, which consists of 11 related cytokines known for enhancing innate immune responses. IL-18 plays a pivotal role in bridging innate and adaptive immunity. It exerts its effects on a wide range of immune cells, including T helper 1 (Th1) cells, macrophages, natural killer (NK) cells, natural killer T (NKT) cells, B cells, dendritic cells (DCs), and even unpolarized T cells, promoting the production of interferon-gamma (IFN-γ), especially in the presence of interleukin-12 (IL-12). In the absence of IL-12, IL-18 can drive the production of type 2 T helper (Th2) cytokines when acting in combination with IL-2, particularly from NK cells, CD4+ NKT cells, and even Th1-committed cells. IL-18 is produced by a variety of both hematopoietic and non-hematopoietic cells, including monocytes, macrophages, keratinocytes, and mesenchymal cells. This cytokine has the potential to activate inflammatory and cytotoxic immune pathways, contributing to the development of autoimmune conditions. Elevated IL-18 levels have been associated with several diseases, such as psoriasis, systemic lupus erythematosus (SLE), hypertension, chronic kidney disease, multiple sclerosis (MS), and COVID-19, often correlating with increased caspase-1 activity. IL-18 signaling activates the phosphatidylinositol-3 kinase (PI3K) pathway, leading to the activation of downstream effectors like mammalian target of rapamycin (mTOR) and the upregulation of anti-apoptotic proteins such as Bcl-xL and Bcl-2. Furthermore, IL-18 enhances the cytotoxic capacity of NK and CD8+ T cells by promoting the release of perforin and the induction of apoptosis in Fas-expressing target cells. It also stimulates IL-4 production, suggesting a possible role in allergic inflammatory responses.

Background

Interleukin-18 (IL-18) is a member of the interleukin-1 (IL-1) cytokine family, which consists of 11 related cytokines known for enhancing innate immune responses. IL-18 plays a pivotal role in bridging innate and adaptive immunity. It exerts its effects on a wide range of immune cells, including T helper 1 (Th1) cells, macrophages, natural killer (NK) cells, natural killer T (NKT) cells, B cells, dendritic cells (DCs), and even unpolarized T cells, promoting the production of interferon-gamma (IFN-γ), especially in the presence of interleukin-12 (IL-12). In the absence of IL-12, IL-18 can drive the production of type 2 T helper (Th2) cytokines when acting in combination with IL-2, particularly from NK cells, CD4+ NKT cells, and even Th1-committed cells. IL-18 is produced by a variety of both hematopoietic and non-hematopoietic cells, including monocytes, macrophages, keratinocytes, and mesenchymal cells. This cytokine has the potential to activate inflammatory and cytotoxic immune pathways, contributing to the development of autoimmune conditions. Elevated IL-18 levels have been associated with several diseases, such as psoriasis, systemic lupus erythematosus (SLE), hypertension, chronic kidney disease, multiple sclerosis (MS), and COVID-19, often correlating with increased caspase-1 activity. IL-18 signaling activates the phosphatidylinositol-3 kinase (PI3K) pathway, leading to the activation of downstream effectors like mammalian target of rapamycin (mTOR) and the upregulation of anti-apoptotic proteins such as Bcl-xL and Bcl-2. Furthermore, IL-18 enhances the cytotoxic capacity of NK and CD8+ T cells by promoting the release of perforin and the induction of apoptosis in Fas-expressing target cells. It also stimulates IL-4 production, suggesting a possible role in allergic inflammatory responses.

References
https://pubmed.ncbi.nlm.nih.gov/36032110/

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%