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생물학적 설명

특이성	TAB1 Antibody [M21E14]는 총 TAB1 단백질의 내인성 수준을 감지합니다.
배경	형질전환 성장 인자-β (TGF-β)-활성화 키나아제 1 (TAK1)은 MAPK 키나아제 키나아제 (MAPKKK) 계열의 세린/트레오닌 키나아제입니다. TAK1은 결합 단백질 TAB1, TAB2 및 TAB3와 결합하며 TNFα, IL-1β 및 톨 유사 수용체 리간드에 의해 활성화되어 NF-κB 및 MAPKs 활성화에 필수적인 역할을 합니다. TAB1은 TAK1과 상호작용하는 어댑터 단백질로, 고혈당(HG) 유도 BMM에서 NF-κB 활성화에 중요하며, NF-κB/HIF-1α를 통해 대식세포 활성화 및 해당과정에 영향을 미칩니다. TAB1은 TAK1의 N-말단 키나아제 도메인과 구성적으로 연관되어 있습니다. 과도한 TAB1은 TAK1 키나아제 활성을 향상시켜 NF-κB 신호 전달을 활성화합니다. TAB1 결핍이 TNFα 및 IL-1β 유도 NF-κB 활성화에 미미한 영향을 미치지만, Thr187에서의 TAK1 인산화를 현저히 손상시킵니다. TAK1-TABs 복합체는 NF-κB 활성화에 필수적인 IKKβ를 인산화하며, MAPK 활성화에 중요합니다. TAK1은 이어서 IKK 및 MAPK를 인산화하여 NF-κB 및 AP-1 활성화를 유도합니다. TAK1-TABs 복합체는 선천 면역 및 염증 반응에서 중요한 역할을 합니다.

특이성

TAB1 Antibody [M21E14]는 총 TAB1 단백질의 내인성 수준을 감지합니다.

배경

형질전환 성장 인자-β (TGF-β)-활성화 키나아제 1 (TAK1)은 MAPK 키나아제 키나아제 (MAPKKK) 계열의 세린/트레오닌 키나아제입니다. TAK1은 결합 단백질 TAB1, TAB2 및 TAB3와 결합하며 TNFα, IL-1β 및 톨 유사 수용체 리간드에 의해 활성화되어 NF-κB 및 MAPKs 활성화에 필수적인 역할을 합니다. TAB1은 TAK1과 상호작용하는 어댑터 단백질로, 고혈당(HG) 유도 BMM에서 NF-κB 활성화에 중요하며, NF-κB/HIF-1α를 통해 대식세포 활성화 및 해당과정에 영향을 미칩니다. TAB1은 TAK1의 N-말단 키나아제 도메인과 구성적으로 연관되어 있습니다. 과도한 TAB1은 TAK1 키나아제 활성을 향상시켜 NF-κB 신호 전달을 활성화합니다. TAB1 결핍이 TNFα 및 IL-1β 유도 NF-κB 활성화에 미미한 영향을 미치지만, Thr187에서의 TAK1 인산화를 현저히 손상시킵니다. TAK1-TABs 복합체는 NF-κB 활성화에 필수적인 IKKβ를 인산화하며, MAPK 활성화에 중요합니다. TAK1은 이어서 IKK 및 MAPK를 인산화하여 NF-κB 및 AP-1 활성화를 유도합니다. TAK1-TABs 복합체는 선천 면역 및 염증 반응에서 중요한 역할을 합니다.

사용 정보

응용

WB, IP

희석

WB	IP
1:1000	1:100

반응성

Human, Monkey

출처

Rabbit Monoclonal Antibody

MW

60 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 649. 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

649. 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/33044562/
https://pubmed.ncbi.nlm.nih.gov/33469458/

적용 데이터

WB

Selleck 검증

Lane 1: Hela
Lane 2: Jurkat
Lane 3: Caki