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생물학적 설명

특이성	LIN28B Antibody [A15P17]는 총 LIN28B 단백질의 내인성 수준을 인식합니다.
배경	LIN28은 발생에 중요한 역할을 하는 보존된 RNA 결합 단백질입니다. 이는 mRNA와 상호작용하여 번역 및 안정성을 조절하며, 특정 마이크로RNA(miRNA)의 전구체 또는 1차 전사체에 결합하여 이들의 성숙을 억제할 수도 있습니다. 포유류에서 LIN28 계열은 LIN28A와 LIN28B 두 가지 유전자로 구성되며, 둘 다 골격근 발달, 신경교 형성, 림프구 생성, 생식세포 발달 및 포도당 대사를 포함한 다양한 생리적 과정에 관여합니다. LIN28은 또한 배아줄기(ES) 세포 다능성의 중요한 조절자 역할을 합니다. OCT4, NANOG, SOX2와 같은 다른 다능성 인자와 결합할 때, LIN28A는 체세포를 유도만능줄기세포(iPSC)로 재프로그래밍하는 데 도움을 줄 수 있습니다. 발생 중 LIN28A 및 LIN28B 발현은 엄격하게 조절되며 주로 ES 세포 및 발달 중인 조직에 제한됩니다. 최근 연구에 따르면 LIN28A 및 LIN28B는 다양한 인간 암 및 암세포주에서 상향 조절되며, 종종 let-7 miRNA 수준 감소와 일치합니다. LIN28A 및 LIN28B는 악성 변형을 유도하고, 전이를 촉진하며, 염증을 조절하고, 암 줄기세포 집단을 유지함으로써 종양유전자 역할을 합니다.

특이성

LIN28B Antibody [A15P17]는 총 LIN28B 단백질의 내인성 수준을 인식합니다.

배경

LIN28은 발생에 중요한 역할을 하는 보존된 RNA 결합 단백질입니다. 이는 mRNA와 상호작용하여 번역 및 안정성을 조절하며, 특정 마이크로RNA(miRNA)의 전구체 또는 1차 전사체에 결합하여 이들의 성숙을 억제할 수도 있습니다. 포유류에서 LIN28 계열은 LIN28A와 LIN28B 두 가지 유전자로 구성되며, 둘 다 골격근 발달, 신경교 형성, 림프구 생성, 생식세포 발달 및 포도당 대사를 포함한 다양한 생리적 과정에 관여합니다. LIN28은 또한 배아줄기(ES) 세포 다능성의 중요한 조절자 역할을 합니다. OCT4, NANOG, SOX2와 같은 다른 다능성 인자와 결합할 때, LIN28A는 체세포를 유도만능줄기세포(iPSC)로 재프로그래밍하는 데 도움을 줄 수 있습니다. 발생 중 LIN28A 및 LIN28B 발현은 엄격하게 조절되며 주로 ES 세포 및 발달 중인 조직에 제한됩니다. 최근 연구에 따르면 LIN28A 및 LIN28B는 다양한 인간 암 및 암세포주에서 상향 조절되며, 종종 let-7 miRNA 수준 감소와 일치합니다. LIN28A 및 LIN28B는 악성 변형을 유도하고, 전이를 촉진하며, 염증을 조절하고, 암 줄기세포 집단을 유지함으로써 종양유전자 역할을 합니다.

사용 정보

응용

WB, IP

희석

WB	IP
1:1000	1:100

반응성

Human

출처

Rabbit Monoclonal Antibody

MW

32, 21 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, 60 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 1048. 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, 60 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

1048. 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/30174831/

적용 데이터

WB

Selleck 검증

Lane 1: NTERA-2
Lane 2: Hep G2
Lane 3: Huh7