데이터시트

생물학적 설명

특이성	Integrin β1 Antibody [H6B12]는 총 Integrin β1 단백질의 내인성 수준을 인식합니다.
배경	Integrin β1은 세포 접착, 이동 및 양방향 ECM 신호 전달에 중요한 이종이량체를 형성하기 위해 α- 아단위와 파트너를 이루는 막횡단 수용체입니다. 이의 세포외 도메인 ( βI, 하이브리드, PSI, I-EGF)은 콜라겐/라미닌에 결합하고, 세포질 꼬리는 탈린/빈쿨린을 모집하여 액틴 세포골격을 연결합니다. 이는 외부-내부 (ECM 유발) 및 내부-외부 (세포골격 유도) 신호 전달 동안의 구조적 변화를 통해 생존, 증식 및 분화를 조절하기 위해 FAK/PI3K/MAPK 경로를 활성화합니다. Integrin β1은 화학내성 및 침습성을 향상시켜 암 전이를 촉진하고, 신장/수정체와 같은 기관에서 조직 무결성을 유지하며, 면역 세포 이동을 조절합니다. 또한 손상 후 신경혈관 복구, 줄기세포 매개 재생 및 ECM 리모델링을 통한 섬유증 진행을 유도합니다. β1D (근육 특이적) 및 β1B/C (억제성)와 같은 이형체는 기계적 스트레스 및 세포 주기 정지에 대한 반응을 미세 조정합니다. 성장 인자 수용체와 상호 작용함으로써 생존 촉진 신호를 증폭시켜 암에서 치료 저항성을 촉진합니다. ECM 강성 감지는 상피-중간엽 전이 (EMT) 및 세포 운명 결정에 영향을 미칩니다. 또한 혈관 신생 시 상처 치유 동안 잠정적 기질과의 내피 세포 상호 작용을 매개합니다. Integrin β1은 신경계에서 라미닌 결합을 통해 축삭 성장 및 시냅스 가소성을 안내합니다. 조절 이상은 발달 결함, 만성 염증 및 자가면역 질환을 초래합니다.

특이성

Integrin β1 Antibody [H6B12]는 총 Integrin β1 단백질의 내인성 수준을 인식합니다.

배경

Integrin β1은 세포 접착, 이동 및 양방향 ECM 신호 전달에 중요한 이종이량체를 형성하기 위해 α- 아단위와 파트너를 이루는 막횡단 수용체입니다. 이의 세포외 도메인 ( βI, 하이브리드, PSI, I-EGF)은 콜라겐/라미닌에 결합하고, 세포질 꼬리는 탈린/빈쿨린을 모집하여 액틴 세포골격을 연결합니다. 이는 외부-내부 (ECM 유발) 및 내부-외부 (세포골격 유도) 신호 전달 동안의 구조적 변화를 통해 생존, 증식 및 분화를 조절하기 위해 FAK/PI3K/MAPK 경로를 활성화합니다. Integrin β1은 화학내성 및 침습성을 향상시켜 암 전이를 촉진하고, 신장/수정체와 같은 기관에서 조직 무결성을 유지하며, 면역 세포 이동을 조절합니다. 또한 손상 후 신경혈관 복구, 줄기세포 매개 재생 및 ECM 리모델링을 통한 섬유증 진행을 유도합니다. β1D (근육 특이적) 및 β1B/C (억제성)와 같은 이형체는 기계적 스트레스 및 세포 주기 정지에 대한 반응을 미세 조정합니다. 성장 인자 수용체와 상호 작용함으로써 생존 촉진 신호를 증폭시켜 암에서 치료 저항성을 촉진합니다. ECM 강성 감지는 상피-중간엽 전이 (EMT) 및 세포 운명 결정에 영향을 미칩니다. 또한 혈관 신생 시 상처 치유 동안 잠정적 기질과의 내피 세포 상호 작용을 매개합니다. Integrin β1은 신경계에서 라미닌 결합을 통해 축삭 성장 및 시냅스 가소성을 안내합니다. 조절 이상은 발달 결함, 만성 염증 및 자가면역 질환을 초래합니다.

사용 정보

응용

WB, IHC

희석

WB	IHC
1:1000	1:100 - 1:400

반응성

Human

출처

Rabbit Monoclonal Antibody

MW

90 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: 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 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: 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

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/37932738/
https://pubmed.ncbi.nlm.nih.gov/28510180/

적용 데이터

WB

Selleck 검증

Lane 1: HeLa, Lane 2: HT 1080, Lane 3: DLD-1