데이터시트

생물학적 설명

특이성	NCK1 Antibody [M17H23]는 전체 NCK1 단백질의 내인성 수준을 검출합니다. 상동성에 따라 NCK2도 인식할 수 있습니다.
배경	NCK1은 중요한 신경세포 적응 분자로, 시냅스 분자 활동 매개, 액틴 세포골격 조절, 신경 기능 영향에 중요한 역할을 합니다. NCK1/α와 NCK2/β를 포함하는 NCK 적응 단백질 계열의 일부이며, 세포 표면에서의 pTyr 신호 전달 및 액틴 세포골격 조절에 필수적입니다. NCK1은 하나의 SH2 도메인과 세 개의 SH3 도메인으로 구성되어 있으며, 이는 형태 형성 및 전달과 같은 세포 사건에 중요한 클러스터링 및 액틴 중합에 결정적인 역할을 합니다. NCK1은 외측 편도체의 흥분성 뉴런에서 글루타메이트 방출을 조절하여 공포 기억 형성에 관여하며, 장기 공포 조건화 기억에 영향을 미칩니다. 또한 시냅스 구조와 기능 형성에 중요한 역할을 합니다. NCK1 결핍은 해마에서 수상돌기 가시 밀도 감소 및 시냅스후 밀도 (PSD) 두께 증가와 관련이 있습니다. 또한 Rac1/PAK1/MMP2 신호 경로를 통해 자궁경부 편평상피암 (CSCC)에서 혈관신생을 촉진합니다. NCK1의 과발현은 난소암 (OC)의 공격적인 특성 및 불량한 예후와 관련이 있습니다.

특이성

NCK1 Antibody [M17H23]는 전체 NCK1 단백질의 내인성 수준을 검출합니다. 상동성에 따라 NCK2도 인식할 수 있습니다.

배경

NCK1은 중요한 신경세포 적응 분자로, 시냅스 분자 활동 매개, 액틴 세포골격 조절, 신경 기능 영향에 중요한 역할을 합니다. NCK1/α와 NCK2/β를 포함하는 NCK 적응 단백질 계열의 일부이며, 세포 표면에서의 pTyr 신호 전달 및 액틴 세포골격 조절에 필수적입니다. NCK1은 하나의 SH2 도메인과 세 개의 SH3 도메인으로 구성되어 있으며, 이는 형태 형성 및 전달과 같은 세포 사건에 중요한 클러스터링 및 액틴 중합에 결정적인 역할을 합니다. NCK1은 외측 편도체의 흥분성 뉴런에서 글루타메이트 방출을 조절하여 공포 기억 형성에 관여하며, 장기 공포 조건화 기억에 영향을 미칩니다. 또한 시냅스 구조와 기능 형성에 중요한 역할을 합니다. NCK1 결핍은 해마에서 수상돌기 가시 밀도 감소 및 시냅스후 밀도 (PSD) 두께 증가와 관련이 있습니다. 또한 Rac1/PAK1/MMP2 신호 경로를 통해 자궁경부 편평상피암 (CSCC)에서 혈관신생을 촉진합니다. NCK1의 과발현은 난소암 (OC)의 공격적인 특성 및 불량한 예후와 관련이 있습니다.

사용 정보

응용

WB, IP

희석

WB	IP
1:1000	1:50

반응성

Human, Mouse, Rat, Monkey

출처

Rabbit Monoclonal Antibody

MW

47 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, 120 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 572. 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, 120 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

572. 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/36371406/
https://pubmed.ncbi.nlm.nih.gov/36535770/

적용 데이터

WB

Selleck 검증

Lane 1: A10
Lane 2: 293
Lane 3: COS
Lane 4: NIH/3T3