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생물학적 설명

특이성	c-Fos Antibody [M9L15]는 총 c-Fos 단백질의 내인성 수준을 검출합니다.
배경	핵 암유전자 Fos 계열은 c-Fos, FosB, Fos-related antigen 1 (FRA1) 및 Fos-related antigen 2 (FRA2)로 구성됩니다. 단일 이소형으로 존재하는 다른 Fos 단백질과 달리, FosB는 두 가지 변이체를 가집니다: 전체 길이 FosB와 카르복실 말단 101개 아미노산이 없는 FosB2 (Delta FosB)로 알려진 절단형입니다. Fos 단백질은 성장 인자, 사이토카인, 신경전달물질, 폴리펩타이드 호르몬 및 스트레스와 같은 다양한 세포외 신호에 반응하여 빠르고 일시적으로 발현됩니다. 이 단백질들은 Jun 계열 구성원(c-Jun, JunB 및 JunD)과 이합체를 형성하여 전사 인자 활성 단백질-1 (AP-1)을 형성하며, 이는 DNA의 TRE/AP-1 요소에 결합하여 유전자 전사를 조절합니다. Fos 및 Jun 단백질은 이합체화에 필수적인 류신 지퍼 모티프와 DNA 결합을 촉진하는 기본 도메인을 가지고 있습니다. 다양한 Fos/Jun 이종이합체는 AP-1 의존성 유전자 활성화에 다양한 능력을 가집니다. 발현 수준 외에도, 외부 자극에 대한 반응으로 Erk 키나아제에 의한 Fos 단백질의 인산화는 전사 활성을 향상시킬 수 있습니다. 특히, Erk5에 의한 Ser32 및 Thr232에서의 c-Fos 인산화는 안정성과 핵 내 위치를 증가시킵니다. c-Fos, FosB 또는 FRA2의 비정상적인 발현은 세포의 신생물성 변형으로 이어질 수 있습니다.

특이성

c-Fos Antibody [M9L15]는 총 c-Fos 단백질의 내인성 수준을 검출합니다.

배경

핵 암유전자 Fos 계열은 c-Fos, FosB, Fos-related antigen 1 (FRA1) 및 Fos-related antigen 2 (FRA2)로 구성됩니다. 단일 이소형으로 존재하는 다른 Fos 단백질과 달리, FosB는 두 가지 변이체를 가집니다: 전체 길이 FosB와 카르복실 말단 101개 아미노산이 없는 FosB2 (Delta FosB)로 알려진 절단형입니다. Fos 단백질은 성장 인자, 사이토카인, 신경전달물질, 폴리펩타이드 호르몬 및 스트레스와 같은 다양한 세포외 신호에 반응하여 빠르고 일시적으로 발현됩니다. 이 단백질들은 Jun 계열 구성원(c-Jun, JunB 및 JunD)과 이합체를 형성하여 전사 인자 활성 단백질-1 (AP-1)을 형성하며, 이는 DNA의 TRE/AP-1 요소에 결합하여 유전자 전사를 조절합니다. Fos 및 Jun 단백질은 이합체화에 필수적인 류신 지퍼 모티프와 DNA 결합을 촉진하는 기본 도메인을 가지고 있습니다. 다양한 Fos/Jun 이종이합체는 AP-1 의존성 유전자 활성화에 다양한 능력을 가집니다. 발현 수준 외에도, 외부 자극에 대한 반응으로 Erk 키나아제에 의한 Fos 단백질의 인산화는 전사 활성을 향상시킬 수 있습니다. 특히, Erk5에 의한 Ser32 및 Thr232에서의 c-Fos 인산화는 안정성과 핵 내 위치를 증가시킵니다. c-Fos, FosB 또는 FRA2의 비정상적인 발현은 세포의 신생물성 변형으로 이어질 수 있습니다.

사용 정보

응용

WB, FCM, ChIP

희석

WB	FCM	CHIP
1:1000	1:1600 - 1:6400	1:50

반응성

Human, Mouse, Rat

출처

Rabbit Monoclonal Antibody

MW

62 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 928. 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

928. 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/10963134/
https://pubmed.ncbi.nlm.nih.gov/17018293/

적용 데이터

WB

Selleck 검증

Lane 1: HeLa (serum-starved overnight; TPA,4 hours)
Lane 2: HeLa (serum-starved overnight)

SAMPLE

Selleck 검증

Fig.1 人源胰腺癌细胞裂解液WB未看到条带 (细胞未经刺激)