CXCL11/I-TAC Antibody (Mouse mAb) [F19K22]

Application: Reactivity: Mouse

Lane 1: Recombinant Mouse CXCL11 Protein

Experiment Essentials

WB
Recommended SDS-PAGE separating gel concentration: 20%.
Recommended wet transfer conditions: 200 mA, 60 min，Recommended to use 0.22 μm PVDF membrane.

Usage Information

Dilution
WB1:500-1:1000

Application
WB

Reactivity
Mouse

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
11 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: 20%. 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. ( 0.22 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 primary antibody dilution buffer to prepare the primary antibody working liquid (recommended dilution ratio for primary antibody 1:500), 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: 20%. 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. ( 0.22 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 primary antibody dilution buffer to prepare the primary antibody working liquid (recommended dilution ratio for primary antibody 1:500), 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
CXCL11/I-TAC Antibody (Mouse mAb) [F19K22] detects endogenous levels of total CXCL11/I-TAC protein.

Subcellular Location
Secreted

Uniprot ID
Q9JHH5

Clone
F19K22

Synonym(s)
beta-R1, H174, I-TAC, ITAC, SCYB9B

Background
CXCL11, also known as I-TAC or IP-9, is an interferon-inducible CXC chemokine belonging to the ELR-negative CXCL9/10/11 subfamily. It functions as a high-affinity ligand for the chemokine receptor CXCR3, with additional activity at CXCR7 and antagonist properties at CCR5. CXCL11 is produced as a small secreted protein with the conserved N-terminal CXC motif and typical chemokine fold, forming a compact β-sheet–helix structure whose surface residues define selective interactions with CXCR3 and distinguish its receptor binding and signaling profile from those of the related ligands CXCL9 and CXCL10. Subtle differences in the N-loop and 30s/40s loops underlie its particularly strong CXCR3 engagement and biased receptor activation. Gene expression of CXCL11 is strongly induced by interferon-γ and interferon-β and more weakly by interferon-α, with synergistic enhancement by inflammatory stimuli such as LPS, IL-1β, and TNF-α, leading to robust production in monocytes/macrophages, endothelial cells, keratinocytes, astrocytes, fibroblasts, hepatocytes, and other stromal and parenchymal cells during Th1-type responses and endotoxemia. Secreted CXCL11 binds CXCR3 on activated, but not naïve, T cells and on NK cells, triggers G-protein–dependent calcium flux, and drives chemotaxis of activated T lymphocytes in vitro and in vivo, positioning it as a key organizer of effector T-cell recruitment into inflamed tissues, including the central nervous system during experimental autoimmune encephalomyelitis, where CXCL11 expression follows CXCR3 up-regulation and contributes to the accumulation of Th1 cells in lesions. CXCL11–CXCR3 interactions can reshape adaptive responses: epidermal expression of CXCL11 in leishmaniasis skews local immunity toward a Th2-type profile by reducing IL-12 production by dendritic cells in draining lymph nodes, while CXCL11, acting through CXCR3, also supports the differentiation or repolarization of IL-10–high regulatory T-cell subsets, indicating that this ligand can reinforce inflammatory Th1 trafficking or instruct regulatory and Th2-biased outcomes depending on context. CXCL11 is also a natural antagonist for CCR5 and a ligand for CXCR7, expanding its influence to the modulation of leukocyte responsiveness to other chemokine axes and potentially affecting HIV coreceptor usage, angiogenesis, and tissue remodeling. Elevated CXCL11 is associated with a variety of inflammatory and immune-mediated conditions, including CNS inflammation, infectious diseases such as RSV and influenza, allergic and cutaneous disorders where keratinocyte CXCL11 expression tracks with epidermal T-cell infiltrates, and cardiovascular disease where CXCL11, together with CXCL9 and CXCL10, serves as a biomarker candidate for heart failure and left ventricular dysfunction.

Background

CXCL11, also known as I-TAC or IP-9, is an interferon-inducible CXC chemokine belonging to the ELR-negative CXCL9/10/11 subfamily. It functions as a high-affinity ligand for the chemokine receptor CXCR3, with additional activity at CXCR7 and antagonist properties at CCR5. CXCL11 is produced as a small secreted protein with the conserved N-terminal CXC motif and typical chemokine fold, forming a compact β-sheet–helix structure whose surface residues define selective interactions with CXCR3 and distinguish its receptor binding and signaling profile from those of the related ligands CXCL9 and CXCL10. Subtle differences in the N-loop and 30s/40s loops underlie its particularly strong CXCR3 engagement and biased receptor activation. Gene expression of CXCL11 is strongly induced by interferon-γ and interferon-β and more weakly by interferon-α, with synergistic enhancement by inflammatory stimuli such as LPS, IL-1β, and TNF-α, leading to robust production in monocytes/macrophages, endothelial cells, keratinocytes, astrocytes, fibroblasts, hepatocytes, and other stromal and parenchymal cells during Th1-type responses and endotoxemia. Secreted CXCL11 binds CXCR3 on activated, but not naïve, T cells and on NK cells, triggers G-protein–dependent calcium flux, and drives chemotaxis of activated T lymphocytes in vitro and in vivo, positioning it as a key organizer of effector T-cell recruitment into inflamed tissues, including the central nervous system during experimental autoimmune encephalomyelitis, where CXCL11 expression follows CXCR3 up-regulation and contributes to the accumulation of Th1 cells in lesions. CXCL11–CXCR3 interactions can reshape adaptive responses: epidermal expression of CXCL11 in leishmaniasis skews local immunity toward a Th2-type profile by reducing IL-12 production by dendritic cells in draining lymph nodes, while CXCL11, acting through CXCR3, also supports the differentiation or repolarization of IL-10–high regulatory T-cell subsets, indicating that this ligand can reinforce inflammatory Th1 trafficking or instruct regulatory and Th2-biased outcomes depending on context. CXCL11 is also a natural antagonist for CCR5 and a ligand for CXCR7, expanding its influence to the modulation of leukocyte responsiveness to other chemokine axes and potentially affecting HIV coreceptor usage, angiogenesis, and tissue remodeling. Elevated CXCL11 is associated with a variety of inflammatory and immune-mediated conditions, including CNS inflammation, infectious diseases such as RSV and influenza, allergic and cutaneous disorders where keratinocyte CXCL11 expression tracks with epidermal T-cell infiltrates, and cardiovascular disease where CXCL11, together with CXCL9 and CXCL10, serves as a biomarker candidate for heart failure and left ventricular dysfunction.

References
https://pubmed.ncbi.nlm.nih.gov/15273303/ https://pubmed.ncbi.nlm.nih.gov/15322564/

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%