Datenblatt

Biologische Beschreibung

Spezifität	CD226 Antibody [P6A12] erkennt endogene Spiegel des gesamten CD226-Proteins.
Hintergrund	CD226 (DNAM-1) ist ein transmembranes Glykoprotein, das überwiegend auf Immunzellen wie T-Zellen, NK-Zellen und Monozyten exprimiert wird. Es enthält zwei Immunglobulin-V-ähnliche Domänen (D1 und D2) in seiner extrazellulären Region, eine Transmembrandomäne vom Typ I und eine intrazelluläre zytoplasmatische Domäne mit kritischen Phosphorylierungsstellen bei Y322 und S329 beim Menschen. CD226 fungiert als kostimulatorischer Rezeptor, der die Aktivierung von Immunantworten durch Bindung an Liganden wie PVR (CD155) und Nectin-2 auf Zielzellen erleichtert. Seine Aktivierung induziert Signalkaskaden, die LFA-1-Aggregation, ERK- und AKT-Signalwege umfassen und die Zytotoxizität in NK-Zellen und T-Zellen verstärken. Die Bindung von CD226 an PVR trägt zur Vermittlung der Tumorerkennung und der Immunzellzytotoxizität gegen Malignome bei. Seine Expression ist in erschöpften Immunzellen während chronischer Infektionen oder Krebs herunterreguliert, was mit einer eingeschränkten Effektorfunktion korreliert. Die Phosphorylierung von CD226 an kritischen Resten, wie Y319, ist entscheidend für seine funktionelle Aktivität und beeinflusst signifikant Immunantworten, einschließlich derer, die an der Tumorimmunität beteiligt sind. Die Mechanismen, die die Herunterregulierung von CD226 antreiben, einschließlich der Eomesodermin-abhängigen transkriptionellen Regulation und der CD155-vermittelten posttranslationalen Modifikationen, tragen zu seiner Dysregulation bei verschiedenen Krankheiten, insbesondere Krebs, bei.

Spezifität

CD226 Antibody [P6A12] erkennt endogene Spiegel des gesamten CD226-Proteins.

Hintergrund

CD226 (DNAM-1) ist ein transmembranes Glykoprotein, das überwiegend auf Immunzellen wie T-Zellen, NK-Zellen und Monozyten exprimiert wird. Es enthält zwei Immunglobulin-V-ähnliche Domänen (D1 und D2) in seiner extrazellulären Region, eine Transmembrandomäne vom Typ I und eine intrazelluläre zytoplasmatische Domäne mit kritischen Phosphorylierungsstellen bei Y322 und S329 beim Menschen. CD226 fungiert als kostimulatorischer Rezeptor, der die Aktivierung von Immunantworten durch Bindung an Liganden wie PVR (CD155) und Nectin-2 auf Zielzellen erleichtert. Seine Aktivierung induziert Signalkaskaden, die LFA-1-Aggregation, ERK- und AKT-Signalwege umfassen und die Zytotoxizität in NK-Zellen und T-Zellen verstärken. Die Bindung von CD226 an PVR trägt zur Vermittlung der Tumorerkennung und der Immunzellzytotoxizität gegen Malignome bei. Seine Expression ist in erschöpften Immunzellen während chronischer Infektionen oder Krebs herunterreguliert, was mit einer eingeschränkten Effektorfunktion korreliert. Die Phosphorylierung von CD226 an kritischen Resten, wie Y319, ist entscheidend für seine funktionelle Aktivität und beeinflusst signifikant Immunantworten, einschließlich derer, die an der Tumorimmunität beteiligt sind. Die Mechanismen, die die Herunterregulierung von CD226 antreiben, einschließlich der Eomesodermin-abhängigen transkriptionellen Regulation und der CD155-vermittelten posttranslationalen Modifikationen, tragen zu seiner Dysregulation bei verschiedenen Krankheiten, insbesondere Krebs, bei.

Nutzungsinformationen

Anwendung

WB, IP, IHC

Verdünnung

WB	IP	IHC
1:1000	1:30	1:100 - 1:500

Reaktivität

Human

Quelle

Rabbit Monoclonal Antibody

MW

39 kDa

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

Experimentelle Methoden
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 transfer the cells into an EP tube. Wash the cells with ice-cold PBS twice. Add 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.Add 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: 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 5% skim milk powder to prepare the primary antibody working liquid (recommended dilution ratio for primary antibody 1:1000), 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 1389. 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.

Experimentelle Methoden

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 transfer the cells into an EP tube. Wash the cells with ice-cold PBS twice. Add 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.Add 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: 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 5% skim milk powder to prepare the primary antibody working liquid (recommended dilution ratio for primary antibody 1:1000), 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

1389. 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.

Referenzen

https://pubmed.ncbi.nlm.nih.gov/33670993/
https://pubmed.ncbi.nlm.nih.gov/24451371/

Anwendungsdaten

WB

Validiert von Selleck

Lane 1: SUP-M2, Lane 2: HH, Lane 3: Jurkat