데이터시트

생물학적 설명

특이성	PI3 Kinase p110 δ Antibody [H17B15]는 총 PI3 Kinase Class III δ 단백질의 내인성 수준을 인식합니다.
배경	포스포이노시티드 3-인산화효소(PI3K)는 포스파티딜이노시톨(PI), 포스파티딜이노시톨-4-인산(PIP), 포스파티딜이노시톨-4,5-비스인산(PIP2)을 인산화하여 포스파티딜이노시톨-3,4,5-삼인산을 생성하는 효소입니다. 이 과정은 성장 인자와 호르몬에 의해 활성화되어 세포 성장, 세포 주기 진행, 세포 이동 및 생존을 조절합니다. PTEN 효소는 포스파티딜이노시톨-3,4,5-삼인산을 탈인산화하여 이 과정을 역전시키며, 연구에 따르면 PTEN 기능 상실은 다양한 인간 암에서 PI3K 경로의 구성적 활성화를 초래합니다. PI3K는 촉매 소단위(p110)와 조절 소단위로 구성되며, p110α, p110β, p110γ 및 p110δ를 포함한 다양한 촉매 소단위 동형이 존재합니다. p85α 및 p85β 조절 소단위는 p110α, p110β 및 p110δ와 상호작용합니다. p110α 동형을 코딩하는 PIK3CA 유전자의 기능 획득 돌연변이가 많은 인간 암에서 흔히 관찰되는 반면, p110β 또는 p110δ를 코딩하는 유전자에서는 체세포 돌연변이가 발견되지 않았습니다. 널리 발현되는 p110α 및 p110β와 달리 p110δ는 주로 백혈구에서 발견되어 p110δ 경로가 면역 질환 연구의 초점이 되고 있습니다. p110δ 동형은 특정 혈액암의 발생 및 진행에 중요한 역할을 하며, p110δ 선택적 억제제 및 마우스 모델에서의 유전적 비활성화를 사용한 연구는 세포 분화, 성장, 생존, 운동성 및 이노시톨 포스파타아제 PTEN과의 상호작용과 같은 과정에 대한 관여를 강조합니다.

특이성

PI3 Kinase p110 δ Antibody [H17B15]는 총 PI3 Kinase Class III δ 단백질의 내인성 수준을 인식합니다.

배경

포스포이노시티드 3-인산화효소(PI3K)는 포스파티딜이노시톨(PI), 포스파티딜이노시톨-4-인산(PIP), 포스파티딜이노시톨-4,5-비스인산(PIP2)을 인산화하여 포스파티딜이노시톨-3,4,5-삼인산을 생성하는 효소입니다. 이 과정은 성장 인자와 호르몬에 의해 활성화되어 세포 성장, 세포 주기 진행, 세포 이동 및 생존을 조절합니다. PTEN 효소는 포스파티딜이노시톨-3,4,5-삼인산을 탈인산화하여 이 과정을 역전시키며, 연구에 따르면 PTEN 기능 상실은 다양한 인간 암에서 PI3K 경로의 구성적 활성화를 초래합니다. PI3K는 촉매 소단위(p110)와 조절 소단위로 구성되며, p110α, p110β, p110γ 및 p110δ를 포함한 다양한 촉매 소단위 동형이 존재합니다. p85α 및 p85β 조절 소단위는 p110α, p110β 및 p110δ와 상호작용합니다. p110α 동형을 코딩하는 PIK3CA 유전자의 기능 획득 돌연변이가 많은 인간 암에서 흔히 관찰되는 반면, p110β 또는 p110δ를 코딩하는 유전자에서는 체세포 돌연변이가 발견되지 않았습니다. 널리 발현되는 p110α 및 p110β와 달리 p110δ는 주로 백혈구에서 발견되어 p110δ 경로가 면역 질환 연구의 초점이 되고 있습니다. p110δ 동형은 특정 혈액암의 발생 및 진행에 중요한 역할을 하며, p110δ 선택적 억제제 및 마우스 모델에서의 유전적 비활성화를 사용한 연구는 세포 분화, 성장, 생존, 운동성 및 이노시톨 포스파타아제 PTEN과의 상호작용과 같은 과정에 대한 관여를 강조합니다.

사용 정보

응용

WB, IP

희석

WB	IP
1:1000	1:50

반응성

Human, Mouse, Rat

출처

Rabbit Monoclonal Antibody

MW

110 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/Tris-HCL 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/Tris-HCL 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/Tris-HCL 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 1128. 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/Tris-HCL 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/Tris-HCL 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/Tris-HCL 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

1128. 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/12040186/
https://pubmed.ncbi.nlm.nih.gov/23459844/

적용 데이터

WB

Selleck 검증

Lane 1: SW620
Lane 2: HT1080