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특이성	RBAP46/RBAP48 Antibody [P6E20]는 총 RBAP46 및 RBAP48 단백질의 내인성 수준을 인식합니다.
배경	RbAp46 및 RbAp48 (각각 RBBP7 및 RBBP4로도 알려짐)은 염색질 구조의 확립 및 유지에 중요한 역할을 하는 매우 유사한 히스톤 샤페론입니다. 이 단백질은 히스톤 H4의 N-말단 꼬리에 있는 리신 잔기의 ɛ-아미노 그룹에서 아세틸 그룹을 추가하거나 제거하는 데 관여하는 다양한 복합체의 핵심 구성 요소이며, 히스톤 메틸전이효소 복합체의 필수적인 부분입니다. RbAp46 및 RbAp48은 히스톤 H4의 히스톤 폴드 영역에 특이적으로 결합하며 염색질 리모델링, 히스톤 아세틸화 및 탈아세틸화 복합체를 히스톤 기질로 유도하는 데 도움을 주는 것으로 생각됩니다. RbAp46은 B형 히스톤 아세틸전이효소 HAT1의 활성을 특이적으로 결합하고 향상시키며, 이는 DNA 복제 중 히스톤이 뉴클레오솜에 통합되기 전에 히스톤 H4의 리신 잔기 5번과 12번을 아세틸화합니다. 반면에 RbAp48은 염색질 조립 인자-1 (CAF-1) 복합체의 진화적으로 보존된 서브유닛으로, 인간 세포에서 다른 두 서브유닛인 p150 및 p60과 상호 작용합니다. RbAp46 및 RbAp48은 모두 수많은 다른 단백질 복합체에 관여하며, 때로는 동일한 복합체 내에 공존하기도 합니다. 또한 뉴클레오솜 리모델링 인자 (NURF) 복합체, 뉴클레오솜 리모델링 및 탈아세틸화 (NuRD) 복합체, Sin3/HDAC 히스톤 탈아세틸화 복합체에도 존재합니다.

특이성

RBAP46/RBAP48 Antibody [P6E20]는 총 RBAP46 및 RBAP48 단백질의 내인성 수준을 인식합니다.

배경

RbAp46 및 RbAp48 (각각 RBBP7 및 RBBP4로도 알려짐)은 염색질 구조의 확립 및 유지에 중요한 역할을 하는 매우 유사한 히스톤 샤페론입니다. 이 단백질은 히스톤 H4의 N-말단 꼬리에 있는 리신 잔기의 ɛ-아미노 그룹에서 아세틸 그룹을 추가하거나 제거하는 데 관여하는 다양한 복합체의 핵심 구성 요소이며, 히스톤 메틸전이효소 복합체의 필수적인 부분입니다. RbAp46 및 RbAp48은 히스톤 H4의 히스톤 폴드 영역에 특이적으로 결합하며 염색질 리모델링, 히스톤 아세틸화 및 탈아세틸화 복합체를 히스톤 기질로 유도하는 데 도움을 주는 것으로 생각됩니다. RbAp46은 B형 히스톤 아세틸전이효소 HAT1의 활성을 특이적으로 결합하고 향상시키며, 이는 DNA 복제 중 히스톤이 뉴클레오솜에 통합되기 전에 히스톤 H4의 리신 잔기 5번과 12번을 아세틸화합니다. 반면에 RbAp48은 염색질 조립 인자-1 (CAF-1) 복합체의 진화적으로 보존된 서브유닛으로, 인간 세포에서 다른 두 서브유닛인 p150 및 p60과 상호 작용합니다. RbAp46 및 RbAp48은 모두 수많은 다른 단백질 복합체에 관여하며, 때로는 동일한 복합체 내에 공존하기도 합니다. 또한 뉴클레오솜 리모델링 인자 (NURF) 복합체, 뉴클레오솜 리모델링 및 탈아세틸화 (NuRD) 복합체, Sin3/HDAC 히스톤 탈아세틸화 복합체에도 존재합니다.

사용 정보

응용

WB

희석

WB

1:1000

반응성

Human, Mouse, Rat, Monkey

출처

Rabbit Monoclonal Antibody

MW

48 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: 10%. 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 922. 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: 10%. 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

922. 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/18571423/

적용 데이터

WB

Selleck 검증

Lane 1: SH-SY5Y
Lane 2: COS-7
Lane 3: F9
Lane 4: H-4-II-E