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생물학적 설명

특이성	CD226 Antibody [P6A12]는 총 CD226 단백질의 내인성 수준을 인식합니다.
배경	CD226 (DNAM-1)은 T 세포, NK 세포 및 단핵구와 같은 면역 세포에 주로 발현되는 막관통 당단백질입니다. 이는 세포외 영역에 두 개의 면역글로불린 V-유사 도메인(D1 및 D2), 유형 I 막관통 도메인, 그리고 인간에서 Y322 및 S329에 중요한 인산화 부위를 갖는 세포내 세포질 도메인을 포함합니다. CD226은 PVR (CD155) 및 넥틴-2와 같은 리간드와 표적 세포에서 결합하여 면역 반응 활성화를 촉진하는 보조 자극 수용체로 기능합니다. 그 활성화는 LFA-1 응집, ERK 및 AKT 경로를 포함하는 신호 전달 캐스케이드를 유도하여 NK 세포 및 T 세포의 세포 독성을 향상시킵니다. CD226과 PVR의 결합은 종양 인식 및 악성 종양에 대한 면역 세포 독성 매개에 도움이 됩니다. 만성 감염 또는 암 동안 소진된 면역 세포에서 그 발현이 하향 조절되며, 이는 손상된 이펙터 기능과 관련이 있습니다. Y319와 같은 중요한 잔기에서 CD226의 인산화는 기능적 활동에 중요하며 종양 면역을 포함한 면역 반응에 상당한 영향을 미칩니다. Eomesodermin 의존성 전사 조절 및 CD155 매개 번역 후 변형을 포함하여 CD226 하향 조절을 유도하는 메커니즘은 다양한 질병, 특히 암에서 그 조절 이상에 기여합니다.

특이성

CD226 Antibody [P6A12]는 총 CD226 단백질의 내인성 수준을 인식합니다.

배경

CD226 (DNAM-1)은 T 세포, NK 세포 및 단핵구와 같은 면역 세포에 주로 발현되는 막관통 당단백질입니다. 이는 세포외 영역에 두 개의 면역글로불린 V-유사 도메인(D1 및 D2), 유형 I 막관통 도메인, 그리고 인간에서 Y322 및 S329에 중요한 인산화 부위를 갖는 세포내 세포질 도메인을 포함합니다. CD226은 PVR (CD155) 및 넥틴-2와 같은 리간드와 표적 세포에서 결합하여 면역 반응 활성화를 촉진하는 보조 자극 수용체로 기능합니다. 그 활성화는 LFA-1 응집, ERK 및 AKT 경로를 포함하는 신호 전달 캐스케이드를 유도하여 NK 세포 및 T 세포의 세포 독성을 향상시킵니다. CD226과 PVR의 결합은 종양 인식 및 악성 종양에 대한 면역 세포 독성 매개에 도움이 됩니다. 만성 감염 또는 암 동안 소진된 면역 세포에서 그 발현이 하향 조절되며, 이는 손상된 이펙터 기능과 관련이 있습니다. Y319와 같은 중요한 잔기에서 CD226의 인산화는 기능적 활동에 중요하며 종양 면역을 포함한 면역 반응에 상당한 영향을 미칩니다. Eomesodermin 의존성 전사 조절 및 CD155 매개 번역 후 변형을 포함하여 CD226 하향 조절을 유도하는 메커니즘은 다양한 질병, 특히 암에서 그 조절 이상에 기여합니다.

사용 정보

응용

WB, IP, IHC

희석

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

반응성

Human

출처

Rabbit Monoclonal Antibody

MW

39 kDa

보관 완충액

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

보관
(수령일로부터)

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

실험 방법
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.

실험 방법

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.

참조

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

적용 데이터

WB

Selleck 검증

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