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생물학적 설명

특이성	IREB2/IRP2 + Aconitase 1/ACO1 Antibody [A13H1]는 총 IREB2/IRP2 + Aconitase 1/ACO1 단백질의 내인성 수준을 인식합니다.
배경	철 조절 단백질 2 (IRP2) 및 아코니타제 1 (ACO1, IRP1으로도 알려짐)은 철 항상성 및 세포 에너지 대사에 필수적인 세포질 단백질입니다. IRP2는 염색체 15q25.1의 IREB2 유전자에 의해 암호화되며, 주로 세포질 철 수준을 감지하여 표적 mRNA의 철 반응 요소 (IRE)에 결합하여 트랜스페린 수용체 1 (TfR1) 및 페리틴과 같은 철 대사 유전자의 발현을 조절합니다. 이는 철을 대사하는 조직에서 널리 발현되며 철 의존적 분해에 의해 조절됩니다. IREB1 유전자에 의해 암호화되는 ACO1은 높은 철 조건에서 시트르산 회로에서 세포질 아코니타제로 작용하고 낮은 철 상태에서 IRE 함유 mRNA를 조절하기 위해 IRP 유사 형태로 전환되는 이중 기능 효소입니다. 구조적으로 두 단백질 모두 [4Fe-4S] 클러스터의 존재에 의해 조절되며, 이는 그들의 효소적 또는 RNA 결합 활성을 제어합니다. 철 저장, 흡수 및 수출의 균형을 맞추는 이들의 역할은 신경 퇴행, 빈혈 및 산화 스트레스 관련 손상과 같은 질병을 예방하는 데 중요하며, 세포 철 및 대사 항상성을 유지하는 데 있어서 그 중요성을 강조합니다.

특이성

IREB2/IRP2 + Aconitase 1/ACO1 Antibody [A13H1]는 총 IREB2/IRP2 + Aconitase 1/ACO1 단백질의 내인성 수준을 인식합니다.

배경

철 조절 단백질 2 (IRP2) 및 아코니타제 1 (ACO1, IRP1으로도 알려짐)은 철 항상성 및 세포 에너지 대사에 필수적인 세포질 단백질입니다. IRP2는 염색체 15q25.1의 IREB2 유전자에 의해 암호화되며, 주로 세포질 철 수준을 감지하여 표적 mRNA의 철 반응 요소 (IRE)에 결합하여 트랜스페린 수용체 1 (TfR1) 및 페리틴과 같은 철 대사 유전자의 발현을 조절합니다. 이는 철을 대사하는 조직에서 널리 발현되며 철 의존적 분해에 의해 조절됩니다. IREB1 유전자에 의해 암호화되는 ACO1은 높은 철 조건에서 시트르산 회로에서 세포질 아코니타제로 작용하고 낮은 철 상태에서 IRE 함유 mRNA를 조절하기 위해 IRP 유사 형태로 전환되는 이중 기능 효소입니다. 구조적으로 두 단백질 모두 [4Fe-4S] 클러스터의 존재에 의해 조절되며, 이는 그들의 효소적 또는 RNA 결합 활성을 제어합니다. 철 저장, 흡수 및 수출의 균형을 맞추는 이들의 역할은 신경 퇴행, 빈혈 및 산화 스트레스 관련 손상과 같은 질병을 예방하는 데 중요하며, 세포 철 및 대사 항상성을 유지하는 데 있어서 그 중요성을 강조합니다.

사용 정보

응용

WB, FCM

희석

WB	FCM
1:1000 -1: 2000	1:50

반응성

Human, Mouse, Rat

출처

Rabbit Monoclonal Antibody

MW

105 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 1271. 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

1271. 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://www.intechopen.com/chapters/37587
https://pubmed.ncbi.nlm.nih.gov/36059676/

적용 데이터

WB

Selleck 검증

Lane 1: Human fetal liver
Lane 2: Jurkat
Lane 3: Mouse kidney
Lane 4: Mouse heart
Lane 5: Rat heart