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Descripción biológica

Especificidad	CD226 Antibody [P6A12] reconoce los niveles endógenos de proteína CD226 total.
Antecedentes	CD226 (DNAM-1) es una glicoproteína transmembrana expresada predominantemente en células inmunes como las células T, las células NK y los monocitos. Contiene dos dominios tipo V de inmunoglobulina (D1 y D2) en su región extracelular, un dominio transmembrana tipo I y un dominio citoplásmico intracelular con sitios de fosforilación críticos en Y322 y S329 en humanos. CD226 funciona como un receptor coestimulador, facilitando la activación de las respuestas inmunes al unirse a ligandos como PVR (CD155) y nectina-2 en las células diana. Su activación induce cascadas de señalización que involucran la agregación de LFA-1, las vías ERK y AKT, mejorando la citotoxicidad en las células NK y las células T. El compromiso de CD226 con PVR ayuda a mediar el reconocimiento tumoral y la citotoxicidad de las células inmunes contra las malignidades. Su expresión está regulada a la baja en células inmunes agotadas durante infecciones crónicas o cáncer, correlacionándose con una función efectora deteriorada. La fosforilación de CD226 en residuos críticos, como Y319, es crucial para su actividad funcional e impacta significativamente las respuestas inmunes, incluyendo aquellas involucradas en la inmunidad tumoral. Los mecanismos que impulsan la regulación a la baja de CD226, incluida la regulación transcripcional dependiente de Eomesodermina y las modificaciones postraduccionales mediadas por CD155, contribuyen a su desregulación en varias enfermedades, especialmente el cáncer.

Especificidad

CD226 Antibody [P6A12] reconoce los niveles endógenos de proteína CD226 total.

Antecedentes

CD226 (DNAM-1) es una glicoproteína transmembrana expresada predominantemente en células inmunes como las células T, las células NK y los monocitos. Contiene dos dominios tipo V de inmunoglobulina (D1 y D2) en su región extracelular, un dominio transmembrana tipo I y un dominio citoplásmico intracelular con sitios de fosforilación críticos en Y322 y S329 en humanos. CD226 funciona como un receptor coestimulador, facilitando la activación de las respuestas inmunes al unirse a ligandos como PVR (CD155) y nectina-2 en las células diana. Su activación induce cascadas de señalización que involucran la agregación de LFA-1, las vías ERK y AKT, mejorando la citotoxicidad en las células NK y las células T. El compromiso de CD226 con PVR ayuda a mediar el reconocimiento tumoral y la citotoxicidad de las células inmunes contra las malignidades. Su expresión está regulada a la baja en células inmunes agotadas durante infecciones crónicas o cáncer, correlacionándose con una función efectora deteriorada. La fosforilación de CD226 en residuos críticos, como Y319, es crucial para su actividad funcional e impacta significativamente las respuestas inmunes, incluyendo aquellas involucradas en la inmunidad tumoral. Los mecanismos que impulsan la regulación a la baja de CD226, incluida la regulación transcripcional dependiente de Eomesodermina y las modificaciones postraduccionales mediadas por CD155, contribuyen a su desregulación en varias enfermedades, especialmente el cáncer.

Información de uso

Aplicación

WB, IP, IHC

Dilución

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

Reactividad

Human

Fuente

Rabbit Monoclonal Antibody

MW

39 kDa

Tampón de almacenamiento

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

Almacenamiento
(Desde la fecha de recepción)

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

Métodos experimentales
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.

Métodos experimentales

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.

Referencias

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

Datos de aplicación

WB

Validado por Selleck

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