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생물학적 설명

특이성	SREBP1 Antibody [J19N8]는 총 SREBP1 단백질의 내인성 수준을 검출합니다.
배경	스테롤 조절 요소 결합 단백질 1(SREBP1)은 SREBF1 유전자에 의해 암호화되는 전사 인자로, 지질 Metabolism 및 항상성에 중요합니다. 이는 SREBP-1a와 SREBP-1c의 두 가지 이소폼으로 존재하며, 서로 다른 프로모터에서 생성되고 조직 특이적인 기능을 나타냅니다. SREBP1은 지방산 및 콜레스테롤 생합성에 관련된 유전자(Fasn, Acac, Scd1 및 Pklr 포함)를 조절하며, 주로 간, 지방 조직 및 근육에서 발현됩니다. 인슐린 및 포도당과 같은 영양 및 호르몬 신호는 SREBP1 발현, 특히 탄수화물 Metabolism 동안 SREBP-1c 이소폼의 발현을 상향 조절합니다. 비활성 ER 결합 전구체로 합성된 SREBP1은 낮은 스테롤 수치 또는 인슐린 신호 전달 하에서 단백질 분해 절단 시 활성화되어 핵으로 이동하여 유전자 프로모터의 스테롤 조절 요소(SREs)에 결합합니다. 구조적으로 SREBP1은 DNA 결합을 위한 N-말단 기본 헬릭스-루프-헬릭스(bHLH) 도메인, 스테롤 조절 요소 결합 도메인 및 보조 인자를 모집하는 C-말단 전사 활성화 도메인을 포함합니다. 이 구조는 SREBP1이 해당 및 지방 생성 유전자 발현을 조율하고 영양분 가용성과 대사 조절을 연결하며, 기능 이상은 인슐린 저항성, 당뇨병 및 기타 대사 질환과 관련이 있습니다.

특이성

SREBP1 Antibody [J19N8]는 총 SREBP1 단백질의 내인성 수준을 검출합니다.

배경

스테롤 조절 요소 결합 단백질 1(SREBP1)은 SREBF1 유전자에 의해 암호화되는 전사 인자로, 지질 Metabolism 및 항상성에 중요합니다. 이는 SREBP-1a와 SREBP-1c의 두 가지 이소폼으로 존재하며, 서로 다른 프로모터에서 생성되고 조직 특이적인 기능을 나타냅니다. SREBP1은 지방산 및 콜레스테롤 생합성에 관련된 유전자(Fasn, Acac, Scd1 및 Pklr 포함)를 조절하며, 주로 간, 지방 조직 및 근육에서 발현됩니다. 인슐린 및 포도당과 같은 영양 및 호르몬 신호는 SREBP1 발현, 특히 탄수화물 Metabolism 동안 SREBP-1c 이소폼의 발현을 상향 조절합니다. 비활성 ER 결합 전구체로 합성된 SREBP1은 낮은 스테롤 수치 또는 인슐린 신호 전달 하에서 단백질 분해 절단 시 활성화되어 핵으로 이동하여 유전자 프로모터의 스테롤 조절 요소(SREs)에 결합합니다. 구조적으로 SREBP1은 DNA 결합을 위한 N-말단 기본 헬릭스-루프-헬릭스(bHLH) 도메인, 스테롤 조절 요소 결합 도메인 및 보조 인자를 모집하는 C-말단 전사 활성화 도메인을 포함합니다. 이 구조는 SREBP1이 해당 및 지방 생성 유전자 발현을 조율하고 영양분 가용성과 대사 조절을 연결하며, 기능 이상은 인슐린 저항성, 당뇨병 및 기타 대사 질환과 관련이 있습니다.

사용 정보

응용

WB

희석

WB

1:200

반응성

Human

출처

Mouse Monoclonal Antibody

MW

122 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 1370. 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. (Exposure time of at least 60s is recommended)

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

1370. 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. (Exposure time of at least 60s is recommended)

참조

https://pubmed.ncbi.nlm.nih.gov/24398675/
https://pubmed.ncbi.nlm.nih.gov/18654640/

적용 데이터

WB

Selleck 검증

Lane 1: HeLa