Human Dectin-1/CLEC7A Antibody [J4K7]

Application: Reactivity:Human

Usage Information

Dilution
IF1:40-1:125 FCM1:400

Application
IF, FCM

Reactivity
Human

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

Positive Control	Mature human dendritic cells
Negative Control

Experimental Methods

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. Permeabilization 1.Add a detergent such as 0.1–0.3% Triton X-100 to the sample and incubate at room temperature for 10–20 minutes. (Note: This step is only required for intracellular antigens. For antigens expressed on the cell membrane, this step is unnecessary.) 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.

Permeabilization

1.Add a detergent such as 0.1–0.3% Triton X-100 to the sample and incubate at room temperature for 10–20 minutes.

(Note: This step is only required for intracellular antigens. For antigens expressed on the cell membrane, this step is unnecessary.)

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
Human Dectin-1/CLEC7A Antibody [J4K7] detects endogenous levels of total Dectin-1/CLEC7A protein.

Subcellular Location
Cell membrane, Cytoplasm, Membrane

Uniprot ID
Q9BXN2

Clone
J4K7

Synonym
Beta-glucan receptor; BGR; CD369; CLEC7A; CLECSF12; CLECSF12DC-associated C-type lectin 1; superfamilymember 12; C-type lectin domain family 7 member A

Background
Human Dectin-1 (CLEC7A) is a type II transmembrane C-type lectin receptor (CLR) predominantly expressed on myeloid cells, including monocytes, macrophages, dendritic cells, and neutrophils, and serves as the principal pattern recognition receptor for fungal β-glucans in innate immunity. It comprises a single extracellular carbohydrate recognition domain (CRD) lacking a stalk in the short isoform, with a shallow, solvent-exposed binding groove formed by residues W221, N242, and E248 that accommodates β-1,3/β-1,6-glucan polymers from yeast and mycobacterial cell walls through Ca²⁺-independent, lectin-like interactions supported by hydrophobic stacking and hydrogen bonding. The receptor contains a single transmembrane helix and a short cytoplasmic tail bearing an unconventional hemITAM motif (E²YSEI) instead of classical ITAM or ITIM motifs. Upon ligand engagement, Dectin-1 clusters within cholesterol-rich membrane nanodomains, enabling recruitment of Src family kinases (SFKs) to phosphorylate the hemITAM tyrosine, which in turn recruits Syk kinase via its dual SH2 domains. This leads to the docking of the CARD9/Bcl10/MALT1 complex and subsequent activation of NF-κB p65, driving the production of proinflammatory cytokines such as IL-12, IL-23, IL-6, and TNF-α. Co-localization with TLR2 synergistically enhances MAPK signaling (p38, ERK, JNK) and reactive oxygen species (ROS) generation via NOX2. The longer Dectin-1 isoform, which includes a stalk region, allows for zymosan phagocytosis by facilitating CRD multivalency and effective fungal engulfment. This Syk–CARD9–NF-κB signaling axis orchestrates Th17-mediated immunity against pathogens like Candida and Aspergillus, while O-glycosylation (T105 sialyl-core1) of the stalk region paradoxically acts as a counter-ligand for platelet CLEC-2, thereby suppressing thrombosis during infection.

Background

Human Dectin-1 (CLEC7A) is a type II transmembrane C-type lectin receptor (CLR) predominantly expressed on myeloid cells, including monocytes, macrophages, dendritic cells, and neutrophils, and serves as the principal pattern recognition receptor for fungal β-glucans in innate immunity. It comprises a single extracellular carbohydrate recognition domain (CRD) lacking a stalk in the short isoform, with a shallow, solvent-exposed binding groove formed by residues W221, N242, and E248 that accommodates β-1,3/β-1,6-glucan polymers from yeast and mycobacterial cell walls through Ca²⁺-independent, lectin-like interactions supported by hydrophobic stacking and hydrogen bonding. The receptor contains a single transmembrane helix and a short cytoplasmic tail bearing an unconventional hemITAM motif (E²YSEI) instead of classical ITAM or ITIM motifs. Upon ligand engagement, Dectin-1 clusters within cholesterol-rich membrane nanodomains, enabling recruitment of Src family kinases (SFKs) to phosphorylate the hemITAM tyrosine, which in turn recruits Syk kinase via its dual SH2 domains. This leads to the docking of the CARD9/Bcl10/MALT1 complex and subsequent activation of NF-κB p65, driving the production of proinflammatory cytokines such as IL-12, IL-23, IL-6, and TNF-α. Co-localization with TLR2 synergistically enhances MAPK signaling (p38, ERK, JNK) and reactive oxygen species (ROS) generation via NOX2. The longer Dectin-1 isoform, which includes a stalk region, allows for zymosan phagocytosis by facilitating CRD multivalency and effective fungal engulfment. This Syk–CARD9–NF-κB signaling axis orchestrates Th17-mediated immunity against pathogens like Candida and Aspergillus, while O-glycosylation (T105 sialyl-core1) of the stalk region paradoxically acts as a counter-ligand for platelet CLEC-2, thereby suppressing thrombosis during infection.

References
https://pubmed.ncbi.nlm.nih.gov/35237264/ https://pubmed.ncbi.nlm.nih.gov/33588306/

Protein Size	PVDF Transfer Membrane Pore Size
< 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

Tech Support

Handling Instructions

Tel: +1-832-582-8158 Ext:3

If you have any other enquiries, please leave a message.

Protein Size	Separating Gel Percentage
4-40	20%
12-45	15%
10-70	12.5%
15-100	10%
25-100	8%
60-210	5%