데이터시트

생물학적 설명

특이성	Phospho-Rpb1 CTD (Ser7) Antibody [C17G21]는 카복시 말단 도메인(CTD) 헵타펩타이드 반복 [Tyr1, Ser2, Pro3, Thr4, Ser5, Pro6, Ser7]이 Ser7에서 인산화된 경우에만 Rpb1 단백질의 내인성 수준을 인식합니다.
배경	RNA 중합효소 II(RNAPII)는 DNA 의존적 RNA 중합효소로 기능하는 크고 다중 서브유닛 효소이며, 4개의 리보핵산 삼인산을 기질로 이용하여 DNA를 RNA로 전사하는 것을 촉진합니다. Rpb1 또는 POLR2A로 알려진 가장 큰 서브유닛은 고유한 헵타펩타이드 서열(Tyr1, Ser2, Pro3, Thr4, Ser5, Pro6, Ser7)을 특징으로 하며, 이는 카복시 말단 도메인(CTD)에서 최대 52회 반복됩니다. 이 헵타펩타이드 반복은 RNAPII 복합체의 활성을 조절하는 데 중요한 역할을 하는 다양한 번역 후 변형의 대상입니다. 활성 전사 동안 CTD의 인산화는 전사를 크로마틴 리모델링 및 RNA 처리와 연결하는 데 필수적입니다. 이 변형은 전사되는 유전자로 크로마틴 변형 효소와 RNA 처리 단백질의 모집을 제어합니다. 처음에는 RNAPII가 전사 개시 단계에서 저인산화된 CTD를 가지고 있으며, DNA 결합 전사 인자 및 Mediator 복합체와의 상호작용을 통해 유전자 프로모터로 향하게 됩니다. Ser7의 인산화는 작은 핵 RNA(snRNA) 유전자의 효과적인 전사에 특히 중요합니다. 전사 초기 단계에서 CDK7에 의한 Ser7의 인산화는 RPAP2 모집을 돕고, 이는 이어서 Ser5를 탈인산화합니다. 이 과정은 Ser2/Ser7의 이중 인산화 패턴을 초래하여 Integrator 복합체의 결합을 향상시켜 신생 snRNA 전사체의 처리를 촉진합니다.

특이성

Phospho-Rpb1 CTD (Ser7) Antibody [C17G21]는 카복시 말단 도메인(CTD) 헵타펩타이드 반복 [Tyr1, Ser2, Pro3, Thr4, Ser5, Pro6, Ser7]이 Ser7에서 인산화된 경우에만 Rpb1 단백질의 내인성 수준을 인식합니다.

배경

RNA 중합효소 II(RNAPII)는 DNA 의존적 RNA 중합효소로 기능하는 크고 다중 서브유닛 효소이며, 4개의 리보핵산 삼인산을 기질로 이용하여 DNA를 RNA로 전사하는 것을 촉진합니다. Rpb1 또는 POLR2A로 알려진 가장 큰 서브유닛은 고유한 헵타펩타이드 서열(Tyr1, Ser2, Pro3, Thr4, Ser5, Pro6, Ser7)을 특징으로 하며, 이는 카복시 말단 도메인(CTD)에서 최대 52회 반복됩니다. 이 헵타펩타이드 반복은 RNAPII 복합체의 활성을 조절하는 데 중요한 역할을 하는 다양한 번역 후 변형의 대상입니다. 활성 전사 동안 CTD의 인산화는 전사를 크로마틴 리모델링 및 RNA 처리와 연결하는 데 필수적입니다. 이 변형은 전사되는 유전자로 크로마틴 변형 효소와 RNA 처리 단백질의 모집을 제어합니다. 처음에는 RNAPII가 전사 개시 단계에서 저인산화된 CTD를 가지고 있으며, DNA 결합 전사 인자 및 Mediator 복합체와의 상호작용을 통해 유전자 프로모터로 향하게 됩니다. Ser7의 인산화는 작은 핵 RNA(snRNA) 유전자의 효과적인 전사에 특히 중요합니다. 전사 초기 단계에서 CDK7에 의한 Ser7의 인산화는 RPAP2 모집을 돕고, 이는 이어서 Ser5를 탈인산화합니다. 이 과정은 Ser2/Ser7의 이중 인산화 패턴을 초래하여 Integrator 복합체의 결합을 향상시켜 신생 snRNA 전사체의 처리를 촉진합니다.

사용 정보

응용

WB, IP, ChIP

희석

WB	IP	CHIP
1:1000	1:100	1:50

반응성

Human, Mouse, Rat, Monkey

출처

Rabbit Monoclonal Antibody

MW

250 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, Phosphatase 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, Phosphatase 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, Phosphatase 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: 250 mA, 180 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 ( recommending 5% BSA 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 917. 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, Phosphatase 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, Phosphatase 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, Phosphatase 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: 250 mA, 180 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 ( recommending 5% BSA 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

917. 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/19834511/
https://pubmed.ncbi.nlm.nih.gov/20457598/

적용 데이터

WB

Selleck 검증

Lane 1: Hela
Lane 2: 293T
Lane 3: C2C12
Lane 4: H-4-II-E