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특이성	ITCH Antibody [K4F16]는 총 ITCH 단백질의 내인성 수준을 인식합니다.
배경	ITCH는 HECT 도메인을 포함하는 E3 유비퀴틴 리가아제로, 원래 마우스 아구티 유전자좌의 유전학 연구에서 확인되었으며, 여기서 돌연변이는 털 색깔의 뚜렷한 변화를 초래합니다. non-agouti-lethal 18H로 알려진 특정 아구티 돌연변이는 다른 아구티 돌연변이 마우스에서는 볼 수 없는 면역학적 결함을 유발하는 것으로 주목됩니다. 더 어두운 털 색깔을 유발하는 이 18H 돌연변이는 방사선 유발 염색체 역위에 의해 발생하며, 근위 및 원위 역위 파손점으로부터 각각 18개 및 20개의 염기쌍을 삭제하여 아구티 및 Itch 유전자 모두의 발현을 방해합니다. Itch 유전자는 약 113 kDa의 분자량을 가진 854개의 아미노산으로 구성된 단백질을 암호화합니다. ITCH는 HECT형 계열에 속하는 단량체 E3 유비퀴틴 리가아제로 기능하며, N-말단 Ca2+-의존성 인지질 결합 C2 도메인, 단백질-단백질 상호작용에 관여하는 여러 WW 도메인, 및 C-말단 HECT 도메인을 포함하는 모듈식 구조를 특징으로 합니다. HECT 도메인은 특정 E2 유비퀴틴 접합 효소와 상호작용하며, 유비퀴틴을 표적 단백질로 전달하기 전에 유비퀴틴과 티오에스테르 결합을 형성하는 고도로 보존된 시스테인 잔기를 포함합니다. ITCH는 4개의 WW 도메인과 195번에서 246번 잔기 사이에 있는 독특한 프롤린-풍부 영역(PRR)을 포함하며, 이는 조절 기능에서 중요한 역할을 합니다. ITCH 매개 유비퀴틴화는 면역 신호 경로를 넘어 Hedgehog, Wnt/β-카테닌, Hippo 및 Notch 신호 경로의 주요 조절자를 표적으로 하여 세포 신호 전달에 대한 광범위한 영향을 강조합니다.

특이성

ITCH Antibody [K4F16]는 총 ITCH 단백질의 내인성 수준을 인식합니다.

배경

ITCH는 HECT 도메인을 포함하는 E3 유비퀴틴 리가아제로, 원래 마우스 아구티 유전자좌의 유전학 연구에서 확인되었으며, 여기서 돌연변이는 털 색깔의 뚜렷한 변화를 초래합니다. non-agouti-lethal 18H로 알려진 특정 아구티 돌연변이는 다른 아구티 돌연변이 마우스에서는 볼 수 없는 면역학적 결함을 유발하는 것으로 주목됩니다. 더 어두운 털 색깔을 유발하는 이 18H 돌연변이는 방사선 유발 염색체 역위에 의해 발생하며, 근위 및 원위 역위 파손점으로부터 각각 18개 및 20개의 염기쌍을 삭제하여 아구티 및 Itch 유전자 모두의 발현을 방해합니다. Itch 유전자는 약 113 kDa의 분자량을 가진 854개의 아미노산으로 구성된 단백질을 암호화합니다. ITCH는 HECT형 계열에 속하는 단량체 E3 유비퀴틴 리가아제로 기능하며, N-말단 Ca2+-의존성 인지질 결합 C2 도메인, 단백질-단백질 상호작용에 관여하는 여러 WW 도메인, 및 C-말단 HECT 도메인을 포함하는 모듈식 구조를 특징으로 합니다. HECT 도메인은 특정 E2 유비퀴틴 접합 효소와 상호작용하며, 유비퀴틴을 표적 단백질로 전달하기 전에 유비퀴틴과 티오에스테르 결합을 형성하는 고도로 보존된 시스테인 잔기를 포함합니다. ITCH는 4개의 WW 도메인과 195번에서 246번 잔기 사이에 있는 독특한 프롤린-풍부 영역(PRR)을 포함하며, 이는 조절 기능에서 중요한 역할을 합니다. ITCH 매개 유비퀴틴화는 면역 신호 경로를 넘어 Hedgehog, Wnt/β-카테닌, Hippo 및 Notch 신호 경로의 주요 조절자를 표적으로 하여 세포 신호 전달에 대한 광범위한 영향을 강조합니다.

사용 정보

응용

WB, IP

희석

WB	IP
1:1000	1:200

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/SDS/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/SDS/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/SDS/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 816. 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/SDS/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/SDS/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/SDS/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

816. 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/18552861/

적용 데이터

WB

Selleck 검증

Lane 1: Ramos
Lane 2: Raji
Lane 3: NIH/3T3