E.coli LPS Antibody [M15H2] | Primärantikörper

Anwendung: Reaktivität:Escherichia coli

Nutzungsinformationen

Verdünnung
IF1:200

Anwendung
IF, ELISA

Reaktivität
Escherichia coli

Quelle
Mouse Monoclonal Antibody

Lagerpuffer
PBS, pH 7.2+50% Glycerol+0.05% BSA+0.01% NaN3

Lagerung (ab dem Datum des Erhalts)
-20°C (avoid freeze-thaw cycles), 2 years

Positivkontrolle	Mouse hepatocytes
Negativkontrolle

Experimentelle Methoden

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

Biologische Beschreibung

Spezifität
E.coli LPS Antibody [M15H2] specifically detects E.coli LPS.

Klon
M15H2

Synonym
Escherichia coli LPS

Hintergrund
E. coli LPS (Lipopolysaccharide) constitutes the primary structural component and potent endotoxin of the Gram-negative bacterial outer membrane, comprising Lipid A, core oligosaccharide, and O-antigen that collectively maintain membrane integrity while serving as the most powerful immunostimulatory molecule known. Lipid A anchors as a bis-phosphorylated β-1',6-glucosamine disaccharide acylated with six fatty acid chains forming the toxic moiety recognized by host TLR4-MD-2-CD14 receptor complex, while the core region links via Kdo-heptose sugars bearing charged phosphate/ethanolamine groups for membrane stabilization, and the highly variable O-antigen polysaccharide chain of repeating epitopes confers serotype specificity and immune evasion through phagocytosis/complement resistance creating "smooth" bacterial morphology. LPS forms an essential permeability barrier protecting against bile salts, antibiotics, and cationic peptides while triggering catastrophic innate immune activation through MyD88/TRIF-dependent NF-κB and IRF3 pathways driving massive TNF-α, IL-1β, IL-6 cytokine storms that precipitate septic shock, disseminated intravascular coagulation, and multi-organ failure during Gram-negative bacteremia; chronic low-grade endotoxemia from gut translocation contributes to atherosclerosis, neurodegenerative diseases, and metabolic syndrome via sustained vascular inflammation, positioning LPS as both indispensable bacterial structural element and humanity's most dangerous microbial toxin.

Hintergrund

E. coli LPS (Lipopolysaccharide) constitutes the primary structural component and potent endotoxin of the Gram-negative bacterial outer membrane, comprising Lipid A, core oligosaccharide, and O-antigen that collectively maintain membrane integrity while serving as the most powerful immunostimulatory molecule known. Lipid A anchors as a bis-phosphorylated β-1',6-glucosamine disaccharide acylated with six fatty acid chains forming the toxic moiety recognized by host TLR4-MD-2-CD14 receptor complex, while the core region links via Kdo-heptose sugars bearing charged phosphate/ethanolamine groups for membrane stabilization, and the highly variable O-antigen polysaccharide chain of repeating epitopes confers serotype specificity and immune evasion through phagocytosis/complement resistance creating "smooth" bacterial morphology. LPS forms an essential permeability barrier protecting against bile salts, antibiotics, and cationic peptides while triggering catastrophic innate immune activation through MyD88/TRIF-dependent NF-κB and IRF3 pathways driving massive TNF-α, IL-1β, IL-6 cytokine storms that precipitate septic shock, disseminated intravascular coagulation, and multi-organ failure during Gram-negative bacteremia; chronic low-grade endotoxemia from gut translocation contributes to atherosclerosis, neurodegenerative diseases, and metabolic syndrome via sustained vascular inflammation, positioning LPS as both indispensable bacterial structural element and humanity's most dangerous microbial toxin.

Referenzen
https://pubmed.ncbi.nlm.nih.gov/23372159/ https://pubmed.ncbi.nlm.nih.gov/30066669/

Proteingröße	PVDF-Transfermembran Porengröße
< 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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Proteingröße	Trenngel-Prozentsatz
4-40	20%
12-45	15%
10-70	12.5%
15-100	10%
25-100	8%
60-210	5%