Datasheet

Biologische Beschrijving

Specificiteit	SSEA4 Antibody [E20H6] detecteert endogene niveaus van totaal SSEA4-eiwit.
Achtergrond	SSEA4 (Stage-Specific Embryonic Antigen 4) is een gesialyleerd hexasaccharide glycolipide-epitoop (Neu5Acα2-3Galβ1-3GalNAcβ1-3Galα1-4Galβ1-4Glc-Cer) dat wordt aangetroffen op menselijke embryonale stamcellen (hESC's), geïnduceerde pluripotente stamcellen (iPSC's), teratocarcinoomcellen en kanker stamcellen, maar afwezig is in gedifferentieerde somatische of muizen pluripotente cellen. De koolhydraatkopgroep neemt een hoefijzervormige conformatie aan die wordt herkend door MC813-antilichamen via een netwerk van waterstofbruggen waarbij het terminale siaalzuur (Neu5Ac) carboxylaat met Asp54H/Gly53H en N-acetyl GalNAc hydroxylgroepen met Asp49L betrokken zijn, wat de bruikbaarheid ervan ondersteunt voor de specifieke detectie van pluripotentie-toestanden via flowcytometrie en immunohistochemie. De stijve α-Gal-koppeling en β1-3 glycosidische bindingen van het glycan handhaven een stabiele, uitgerekte conformatie bovenop ceramide-lipidestaarten in plasmamembranen, waar SSEA4 de stamcelcapaciteit actief ondersteunt door Wnt/β-catenine-signalering te moduleren via directe interactie met de LRP6 co-receptor, waardoor ligand-onafhankelijke pathway-activering mogelijk wordt, en door PI3K/Akt te rekruteren via glycan-gemedieerde clustering die de Nanog/Oct4/Sox2-circuiterie essentieel voor zelfvernieuwing in stand houdt. Tijdens differentiatie verwijdert downregulatie van glycosyltransferasen (ST3GAL6, B3GALT5) progressief het terminale siaalzuur, waardoor SSEA4 wordt omgezet in SSEA3 en zijn signaleringscompetentie wordt uitgeschakeld.

Specificiteit

SSEA4 Antibody [E20H6] detecteert endogene niveaus van totaal SSEA4-eiwit.

Achtergrond

SSEA4 (Stage-Specific Embryonic Antigen 4) is een gesialyleerd hexasaccharide glycolipide-epitoop (Neu5Acα2-3Galβ1-3GalNAcβ1-3Galα1-4Galβ1-4Glc-Cer) dat wordt aangetroffen op menselijke embryonale stamcellen (hESC's), geïnduceerde pluripotente stamcellen (iPSC's), teratocarcinoomcellen en kanker stamcellen, maar afwezig is in gedifferentieerde somatische of muizen pluripotente cellen. De koolhydraatkopgroep neemt een hoefijzervormige conformatie aan die wordt herkend door MC813-antilichamen via een netwerk van waterstofbruggen waarbij het terminale siaalzuur (Neu5Ac) carboxylaat met Asp54H/Gly53H en N-acetyl GalNAc hydroxylgroepen met Asp49L betrokken zijn, wat de bruikbaarheid ervan ondersteunt voor de specifieke detectie van pluripotentie-toestanden via flowcytometrie en immunohistochemie. De stijve α-Gal-koppeling en β1-3 glycosidische bindingen van het glycan handhaven een stabiele, uitgerekte conformatie bovenop ceramide-lipidestaarten in plasmamembranen, waar SSEA4 de stamcelcapaciteit actief ondersteunt door Wnt/β-catenine-signalering te moduleren via directe interactie met de LRP6 co-receptor, waardoor ligand-onafhankelijke pathway-activering mogelijk wordt, en door PI3K/Akt te rekruteren via glycan-gemedieerde clustering die de Nanog/Oct4/Sox2-circuiterie essentieel voor zelfvernieuwing in stand houdt. Tijdens differentiatie verwijdert downregulatie van glycosyltransferasen (ST3GAL6, B3GALT5) progressief het terminale siaalzuur, waardoor SSEA4 wordt omgezet in SSEA3 en zijn signaleringscompetentie wordt uitgeschakeld.

Gebruiksinformatie

Toepassing

IF, FCM

Verdunning

IF	FCM
1:100 - 1:400	1:200 - 1:800

Reactiviteit

Human

Bron

Mouse Monoclonal Antibody

MW

Opslagbuffer

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

Opslag
(Vanaf de datum van ontvangst)

-20°C (avoid freeze-thaw cycles), 2 years

Experimentele 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.

Experimentele 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.

Referenties

https://pubmed.ncbi.nlm.nih.gov/6141938/
https://pubmed.ncbi.nlm.nih.gov/31831622/

SSEA4 Antibody [E20H6]

Biologische Beschrijving

Gebruiksinformatie

Referenties

Toepassingsgegevens