CX3CR1 Antibody [G13H18] | Primary Antibodies

Application: Reactivity: Human, Mouse, Rat

Lane 1: THP-1, Lane 2: Hela, Lane 3: SW480, Lane 4: HepG2

Experiment Essentials

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

Usage Information

Dilution
WB1:500-1:1000 IF1:500

Application
WB, IF

Reactivity
Human, Mouse, Rat

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 Observed MW
40 kDa 52 kDa
^*Why do the predicted and actual molecular weights differ? The following reasons may explain differences between the predicted and actual protein molecular weight. Post-translational modifications(e.g., phosphorylation, glycosylation); Splice variants and isoforms; Relative charge; Multimerization.

Positive Control	Mouse brain tissue; Rat brain tissue; THP‑1 cells; HeLa cells; SW480 cells; HepG2 cells; Caco‑2 cells; SH‑SY5Y cells
Negative Control

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. Add protein loading buffer to the 20 μL sample, and keep it on ice for immediate use; or determine the optimal denaturation conditions by boiling the sample at a temperature gradient (e.g., 37°C, 50°C, 70°C, 90°C, and 100°C). Cool the sample on ice and centrifuge for 5 min. 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 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. Add protein loading buffer to the 20 μL sample, and keep it on ice for immediate use; or determine the optimal denaturation conditions by boiling the sample at a temperature gradient (e.g., 37°C, 50°C, 70°C, 90°C, and 100°C). Cool the sample on ice and centrifuge for 5 min.

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 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.

IF
Experimental Protocol: Sample Preparation 1. Adherent Cells: Place a clean, sterile coverslip in a culture dish. Once the cells grow to near confluence as a monolayer, remove the coverslip for further use. 2. Suspension Cells: Seed the cells onto a clean, sterile slide coated with poly-L-lysine. 3. Frozen Sections: Allow the slide to thaw at room temperature. Wash it with pure water or PBS for 2 times, 3 minutes each time. 4. Paraffin Sections: Deparaffinization and rehydration. Wash the slide with pure water or PBS for 3 times, 3 minutes each time. Then perform antigen retrieval. Fixation 1. Fix the cell coverslips/spots or tissue sections at room temperature using a fixative such as 4% paraformaldehyde (4% PFA) for 10-15 minutes. 2. Wash the sample with PBS for 3 times, 3 minutes each time. Blocking Add blocking solution and incubate at room temperature for at least 1 hour. (Common blocking solutions include: serum from the same source as the secondary antibody, BSA, or goat serum.) Note: Ensure the sample remains moist during and after the blocking step to prevent drying, which can lead to high background. Immunofluorescence Staining (Day 1) 1. Remove the blocking solution and add the diluted primary antibody. 2. Incubate the sample in a humidified chamber at 4°C overnight. Immunofluorescence Staining (Day 2) 1. Remove the primary antibody and wash with PBST for 3 times, 5 minutes each time. 2. Add the diluted fluorescent secondary antibody and incubate in the dark at 4°C for 1–2 hours. 3. Remove the secondary antibody and wash with PBST for 3 times, 5 minutes each time. 4. Add diluted DAPI and incubate at room temperature in the dark for 5–10 minutes. 5. Wash with PBST for 3 times, 5 minutes each time. Mounting 1. Mount the sample with an anti-fade mounting medium. 2. Allow the slide to dry at room temperature overnight in the dark. 3. Store the slide in a slide storage box at 4°C, protected from light.

IF

Experimental Protocol:

Sample Preparation

1. Adherent Cells: Place a clean, sterile coverslip in a culture dish. Once the cells grow to near confluence as a monolayer, remove the coverslip for further use.

2. Suspension Cells: Seed the cells onto a clean, sterile slide coated with poly-L-lysine.

3. Frozen Sections: Allow the slide to thaw at room temperature. Wash it with pure water or PBS for 2 times, 3 minutes each time.

4. Paraffin Sections: Deparaffinization and rehydration. Wash the slide with pure water or PBS for 3 times, 3 minutes each time. Then perform antigen retrieval.

Fixation

1. Fix the cell coverslips/spots or tissue sections at room temperature using a fixative such as 4% paraformaldehyde (4% PFA) for 10-15 minutes.

2. Wash the sample with PBS for 3 times, 3 minutes each time.

Blocking

Add blocking solution and incubate at room temperature for at least 1 hour. (Common blocking solutions include: serum from the same source as the secondary antibody, BSA, or goat serum.)

Note: Ensure the sample remains moist during and after the blocking step to prevent drying, which can lead to high background.

Immunofluorescence Staining (Day 1)

1. Remove the blocking solution and add the diluted primary antibody.

2. Incubate the sample in a humidified chamber at 4°C overnight.

Immunofluorescence Staining (Day 2)

1. Remove the primary antibody and wash with PBST for 3 times, 5 minutes each time.

2. Add the diluted fluorescent secondary antibody and incubate in the dark at 4°C for 1–2 hours.

3. Remove the secondary antibody and wash with PBST for 3 times, 5 minutes each time.

4. Add diluted DAPI and incubate at room temperature in the dark for 5–10 minutes.

5. Wash with PBST for 3 times, 5 minutes each time.

Mounting

1. Mount the sample with an anti-fade mounting medium.

2. Allow the slide to dry at room temperature overnight in the dark.

3. Store the slide in a slide storage box at 4°C, protected from light.

Datasheet & SDS

Biological Description

Specificity
CX3CR1 Antibody (Rabbit mAb) [G13H18] detects endogenous levels of total CX3CR1 protein.

Subcellular Location
Cell membrane, Membrane

Uniprot ID
P49238

Clone
G13H18

Synonym(s)
Beta chemokine receptor-like 1, C-X3-C CKR-1, CMK-BRL-1, CMK-BRL1, Cx3c, CX3C chemokine receptor 1, V28, CCRL1, CMKBRL1, CMKDR1, CX3CR1, GPR13, GPRV28, Rbs11, V28

Background
CX3CR1 is a seven‑transmembrane G protein–coupled chemokine receptor of the CX3C family that serves as the cognate receptor for fractalkine/CX3CL1 and is expressed on selected immune and parenchymal cell populations, including monocytes, macrophages, microglia, subsets of CD8⁺ T cells, NK cells, and smooth muscle cells. The receptor binds both membrane‑anchored and soluble CX3CL1, with the N‑terminal chemokine domain of CX3CL1 engaging the extracellular loops of CX3CR1, while the transmembrane helices and intracellular loops couple to heterotrimeric G proteins and downstream signaling modules typical of chemokine GPCRs. CX3CR1 activation by membrane‑bound CX3CL1 promotes firm adhesion of CX3CR1‑positive leukocytes to endothelial or epithelial cells under flow, integrating chemokine receptor signaling with integrin activation and cytoskeletal rearrangements to stabilize immune cell–tissue contacts at sites of inflammation or in steady‑state surveillance niches. Soluble CX3CL1 binding to CX3CR1 triggers chemotactic signaling through Gαi‑dependent pathways, leading to inhibition of adenylyl cyclase, activation of PI3K and small GTPases, and reorganization of the actin cytoskeleton that supports directed migration of monocytes, T cells, NK cells, and other CX3CR1‑expressing subsets toward fractalkine gradients. CX3CL1–CX3CR1 engagement also activates survival and activation pathways in CX3CR1⁺ cells, including PI3K–Akt and ERK cascades, and influences expression of cytokines, cytotoxic mediators, and adhesion molecules, linking this axis to cell retention, effector differentiation, and resistance to apoptosis in tissue‑resident and circulating immune populations. In the nervous system, CX3CR1 on microglia and neuron‑associated macrophages binds neuronal CX3CL1 and participates in neuron–glia communication that regulates microglial activation state, synaptic pruning, and inflammatory responses to injury or neurodegeneration. Across inflammatory and infectious contexts, the CX3CL1–CX3CR1 axis coordinates leukocyte recruitment, adhesion, and positioning within tissues and contributes to the balance between protective immunity and chronic inflammation in diseases such as atherosclerosis, autoimmune disorders, and chronic infections. CX3CR1 also appears on CX3CR1⁺ CD8⁺ T cells enriched for effector and memory‑like features, where CX3CL1 binding supports their tissue homing, retention in inflamed or tumor sites, and participation in antitumor cytotoxic responses, making CX3CR1 a marker of highly functional cytotoxic T‑cell subsets. Genetic variation or dysregulated expression of CX3CR1 associates with altered susceptibility or progression in cardiovascular, neuroinflammatory, and malignant diseases, consistent with its central role in controlling leukocyte trafficking, survival, and effector function through fractalkine‑dependent chemotactic and adhesive signaling.

Background

CX3CR1 is a seven‑transmembrane G protein–coupled chemokine receptor of the CX3C family that serves as the cognate receptor for fractalkine/CX3CL1 and is expressed on selected immune and parenchymal cell populations, including monocytes, macrophages, microglia, subsets of CD8⁺ T cells, NK cells, and smooth muscle cells. The receptor binds both membrane‑anchored and soluble CX3CL1, with the N‑terminal chemokine domain of CX3CL1 engaging the extracellular loops of CX3CR1, while the transmembrane helices and intracellular loops couple to heterotrimeric G proteins and downstream signaling modules typical of chemokine GPCRs. CX3CR1 activation by membrane‑bound CX3CL1 promotes firm adhesion of CX3CR1‑positive leukocytes to endothelial or epithelial cells under flow, integrating chemokine receptor signaling with integrin activation and cytoskeletal rearrangements to stabilize immune cell–tissue contacts at sites of inflammation or in steady‑state surveillance niches. Soluble CX3CL1 binding to CX3CR1 triggers chemotactic signaling through Gαi‑dependent pathways, leading to inhibition of adenylyl cyclase, activation of PI3K and small GTPases, and reorganization of the actin cytoskeleton that supports directed migration of monocytes, T cells, NK cells, and other CX3CR1‑expressing subsets toward fractalkine gradients. CX3CL1–CX3CR1 engagement also activates survival and activation pathways in CX3CR1⁺ cells, including PI3K–Akt and ERK cascades, and influences expression of cytokines, cytotoxic mediators, and adhesion molecules, linking this axis to cell retention, effector differentiation, and resistance to apoptosis in tissue‑resident and circulating immune populations. In the nervous system, CX3CR1 on microglia and neuron‑associated macrophages binds neuronal CX3CL1 and participates in neuron–glia communication that regulates microglial activation state, synaptic pruning, and inflammatory responses to injury or neurodegeneration. Across inflammatory and infectious contexts, the CX3CL1–CX3CR1 axis coordinates leukocyte recruitment, adhesion, and positioning within tissues and contributes to the balance between protective immunity and chronic inflammation in diseases such as atherosclerosis, autoimmune disorders, and chronic infections. CX3CR1 also appears on CX3CR1⁺ CD8⁺ T cells enriched for effector and memory‑like features, where CX3CL1 binding supports their tissue homing, retention in inflamed or tumor sites, and participation in antitumor cytotoxic responses, making CX3CR1 a marker of highly functional cytotoxic T‑cell subsets. Genetic variation or dysregulated expression of CX3CR1 associates with altered susceptibility or progression in cardiovascular, neuroinflammatory, and malignant diseases, consistent with its central role in controlling leukocyte trafficking, survival, and effector function through fractalkine‑dependent chemotactic and adhesive signaling.

References
https://pubmed.ncbi.nlm.nih.gov/33391453/ https://pubmed.ncbi.nlm.nih.gov/39377122/

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%

CX3CR1 Antibody (Rabbit mAb) [G13H18]