데이터시트

생물학적 설명

특이성	SMC2 Antibody [L9G12]는 총 SMC2 단백질의 내인성 수준을 인식합니다. 이 항체는 SMC1, SMC3, SMC4를 포함한 다른 SMC 단백질과 교차 반응하지 않습니다.
배경	응축 복합체의 필수 구성 요소인 SMC2 및 SMC4 단백질은 염색체 응축 및 후기 동안 염색체 압축 유지에 중요한 역할을 하여 반대 극으로의 적절한 분리를 보장합니다. 이들 SMC 단백질은 ABC ATPase 슈퍼패밀리에 속하는 특징적인 이분형 ATPase 도메인을 특징으로 하며, N-말단 워커 A 모티프 뉴클레오타이드 결합 도메인과 C-말단 워커 B 모티프 촉매 도메인으로 구성되어 작동 ATPase를 형성하기 위해 협력합니다. 이 두 ATPase 도메인을 연결하는 것은 중앙 힌지 영역으로 분리된 두 개의 코일-코일 도메인으로, 파트너 SMC 단백질 간의 단백질-단백질 상호작용을 촉진합니다. 응축 복합체의 맥락에서 SMC2와 SMC4는 결합하여 염색질 응축에 필수적인 기능적 ATPase를 형성합니다. 고등 유기체는 각각 SMC2 및 SMC4 서브유닛을 포함하는 두 개의1 응축 복합체(응축 I 및 II)를 가지고 있습니다.

특이성

SMC2 Antibody [L9G12]는 총 SMC2 단백질의 내인성 수준을 인식합니다. 이 항체는 SMC1, SMC3, SMC4를 포함한 다른 SMC 단백질과 교차 반응하지 않습니다.

배경

응축 복합체의 필수 구성 요소인 SMC2 및 SMC4 단백질은 염색체 응축 및 후기 동안 염색체 압축 유지에 중요한 역할을 하여 반대 극으로의 적절한 분리를 보장합니다. 이들 SMC 단백질은 ABC ATPase 슈퍼패밀리에 속하는 특징적인 이분형 ATPase 도메인을 특징으로 하며, N-말단 워커 A 모티프 뉴클레오타이드 결합 도메인과 C-말단 워커 B 모티프 촉매 도메인으로 구성되어 작동 ATPase를 형성하기 위해 협력합니다. 이 두 ATPase 도메인을 연결하는 것은 중앙 힌지 영역으로 분리된 두 개의 코일-코일 도메인으로, 파트너 SMC 단백질 간의 단백질-단백질 상호작용을 촉진합니다. 응축 복합체의 맥락에서 SMC2와 SMC4는 결합하여 염색질 응축에 필수적인 기능적 ATPase를 형성합니다. 고등 유기체는 각각 SMC2 및 SMC4 서브유닛을 포함하는 두 개의1 응축 복합체(응축 I 및 II)를 가지고 있습니다.

사용 정보

응용

WB, IP

희석

WB	IP
1:1000	1:100

반응성

Human, Mouse, Rat, Monkey, Pig, Horse

출처

Rabbit Monoclonal Antibody

MW

140 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/Nuclear 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/Nuclear 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/Nuclear 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: 5%. 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 584. 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/Nuclear 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/Nuclear 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/Nuclear 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: 5%. 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

584. 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/15937217/
https://pubmed.ncbi.nlm.nih.gov/19308696/

적용 데이터

WB

Selleck 검증

Lane 1: hela
Lane 2: c2c12
Lane 3: H-4-ll-e
Lane 4: cos-7