데이터시트

생물학적 설명

특이성	UBE3A Antibody [P7K8]는 총 UBE3A 단백질의 내인성 수준을 인식합니다.
배경	E6AP(E6 Associated Protein)로도 알려진 UBE3A는 E3 유비퀴틴 리가아제이며 HECT(Homologous to the E6 Carboxyl Terminus) 계열 E3 리가아제 중 처음으로 확인된 구성원입니다. 이 단백질은 350개의 잔기로 구성된 고도로 보존된 C-말단 촉매 도메인을 특징으로 합니다. 활성화 또는 손실을 통한 E6AP 기능의 변화는 다양한 인간 질병과 관련이 있습니다. 15q11-13 염색체 영역에 위치한 UBE3A 유전자의 결실, 각인 결함 또는 돌연변이로 인한 E6AP 기능 상실은 신경학적 질환인 안젤만 증후군과 관련이 있습니다. UBE3A의 자연 돌연변이 대부분은 완전한 HECT 도메인이 없는 E6AP의 절단된 형태를 생성하는 결실을 초래합니다. 그러나 유전적 변형의 약 10%는 E6AP 코딩 영역 내의 점 돌연변이입니다. 반대로 UBE3A 유전자의 중복은 일부 자폐 스펙트럼 장애의 기여 요인으로 제안되었습니다. 고위험 인유두종바이러스(HPV16 및 HPV18)에서 유래한 발암성 E6 단백질은 UBE3A를 탈취하여 특정 세포 단백질, 특히 p53의 부적절한 유비퀴틴화를 초래할 수 있으며, 이는 암 발생 맥락에서 중요합니다. 뉴런에서 UBE3A 기능의 방해는 흥분성 시냅스에서 Arc 발현 증가 및 AMPA 수용체(AMPAR) 감소를 초래하여 안젤만 증후군의 신경학적 증상에 잠재적으로 기여할 수 있습니다.

특이성

UBE3A Antibody [P7K8]는 총 UBE3A 단백질의 내인성 수준을 인식합니다.

배경

E6AP(E6 Associated Protein)로도 알려진 UBE3A는 E3 유비퀴틴 리가아제이며 HECT(Homologous to the E6 Carboxyl Terminus) 계열 E3 리가아제 중 처음으로 확인된 구성원입니다. 이 단백질은 350개의 잔기로 구성된 고도로 보존된 C-말단 촉매 도메인을 특징으로 합니다. 활성화 또는 손실을 통한 E6AP 기능의 변화는 다양한 인간 질병과 관련이 있습니다. 15q11-13 염색체 영역에 위치한 UBE3A 유전자의 결실, 각인 결함 또는 돌연변이로 인한 E6AP 기능 상실은 신경학적 질환인 안젤만 증후군과 관련이 있습니다. UBE3A의 자연 돌연변이 대부분은 완전한 HECT 도메인이 없는 E6AP의 절단된 형태를 생성하는 결실을 초래합니다. 그러나 유전적 변형의 약 10%는 E6AP 코딩 영역 내의 점 돌연변이입니다. 반대로 UBE3A 유전자의 중복은 일부 자폐 스펙트럼 장애의 기여 요인으로 제안되었습니다. 고위험 인유두종바이러스(HPV16 및 HPV18)에서 유래한 발암성 E6 단백질은 UBE3A를 탈취하여 특정 세포 단백질, 특히 p53의 부적절한 유비퀴틴화를 초래할 수 있으며, 이는 암 발생 맥락에서 중요합니다. 뉴런에서 UBE3A 기능의 방해는 흥분성 시냅스에서 Arc 발현 증가 및 AMPA 수용체(AMPAR) 감소를 초래하여 안젤만 증후군의 신경학적 증상에 잠재적으로 기여할 수 있습니다.

사용 정보

응용

WB

희석

WB

1:1000

반응성

Human, Mouse, Rat, Monkey

출처

Rabbit Monoclonal Antibody

MW

98 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: 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 965. 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)，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: 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

965. 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/24273172/
https://pubmed.ncbi.nlm.nih.gov/8380895/

적용 데이터

WB

Selleck 검증

Lane 1: K562
Lane 2: NIH/3T3
Lane 3: COS-7
Lane 4: A172