Phospho-p70 S6K (Thr421/Ser424) Antibody [E5J12]

카탈로그 번호 F2333

인쇄

생물학적 설명

특이성

Phospho-p70 S6K (Thr421/Ser424) Antibody [E5J12]는 Thr421 및 Ser424에 각각 인산화된 경우에만 p70 S6 kinase의 내인성 수준을 감지합니다.
배경

Phospho-p70 S6 kinase (Thr421/Ser424)는 카르복시 말단 자가억제 도메인 내에 위치한 트레오닌 421 및 세린 424에서 인산화된 p70S6K 형태를 나타냅니다. ribosomal S6 kinase 1 (S6K1)이라고도 불리는 p70S6K는 AGC kinase 계열에 속하는 70-kDa 세린/트레오닌 kinase이며 PI3K/Akt/mTOR 경로의 핵심 하위 이펙터입니다. 전체 길이 효소는 N-말단 조절 영역, 촉매 kinase 도메인 및 활동을 제어하는 C-말단 자가억제 세그먼트를 포함합니다. p70S6K는 포유류 조직에 널리 발현되며, 섬유아세포, 면역 세포 및 발달 중인 조직을 포함한 대사 활성 및 증식성 세포에서 특히 높은 수준으로 나타납니다. Thr421/Ser424의 인산화는 성장 인자, GM-CSF와 같은 사이토카인 및 유사분열 신호에 대한 반응으로 발생하며, 자가억제를 해제하여 Thr389 및 Thr229에서의 후속 인산화를 허용하고, 이는 완전한 활성화를 위한 중요한 단계입니다. 기능적으로 Thr421/Ser424의 인산화는 p70S6K의 효소 활성 증가에 기여하며, 이는 ribosomal protein S6 및 eIF4B와 같은 기질을 인산화하여 번역 개시, 단백질 합성, 세포 주기 진행, 성장 및 증식을 촉진합니다. 호중구와 같은 조혈 세포에서 GM-CSF 유도 인산화는 mTOR 및 MAPK 의존성 경로 모두에서 신호를 통합하며, 이는 염증, 면역 반응 및 성장 제어에서 S6K 활성화를 위한 핵심 조절 체크포인트로서 Phospho-Thr421/Ser424를 강조합니다.

사용 정보

응용 WB, IHC, ELISA 희석
WB IHC
1:500 - 1:3000 1:100
반응성 Human, Mouse, Rat
출처 Rabbit Monoclonal Antibody MW 59 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
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, Phosphatase Inhibitor Cocktail)，and homogenize the tissue at a low temperature.
2. Adherent cell: Aspirate the culture medium and wash the cells with ice-cold PBS twice. Lyse the cells by adding an appropriate volume of RIPA/NP-40 Lysis Buffer (containing Protease Inhibitor Cocktail, Phosphatase Inhibitor Cocktail) and put the sample on ice for 5 min.
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. Lyse the cells by adding an appropriate volume of RIPA/NP-40 Lysis Buffer (containing Protease Inhibitor Cocktail, Phosphatase Inhibitor Cocktail) and put the sample on ice for 5 min.
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 ( recommending 5% BSA 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 1:500), 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
1. 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.
IHC
Experimental Protocol：
 
Deparaffinization/Rehydration
1. Deparaffinize/hydrate sections:
2. Incubate sections in three washes of xylene for 5 min each.
3. Incubate sections in two washes of 100% ethanol for 10 min each.
4. Incubate sections in two washes of 95% ethanol for 10 min each.
5. Wash sections two times in dH2O for 5 min each.
6.Antigen retrieval: For Citrate: Heat slides in a microwave submersed in 1X citrate unmasking solution until boiling is initiated; continue with 10 min at a sub-boiling temperature (95°-98°C). Cool slides on bench top for 30 min.
 
Staining
1. Wash sections in dH2O three times for 5 min each.
2. Incubate sections in 3% hydrogen peroxide for 10 min.
3. Wash sections in dH2O two times for 5 min each.
4. Wash sections in wash buffer for 5 min.
5. Block each section with 100–400 µl of blocking solution for 1 hr at room temperature.
6. Remove blocking solution and add 100–400 µl primary antibody diluent in to each section. Incubate overnight at 4°C.
7. Remove antibody solution and wash sections with wash buffer three times for 5 min each.
8. Cover section with 1–3 drops HRPas needed. Incubate in a humidified chamber for 30 min at room temperature.
9. Wash sections three times with wash buffer for 5 min each.
10. Add DAB Chromogen Concentrate to DAB Diluent and mix well before use.
11. Apply 100–400 µl DAB to each section and monitor closely. 1–10 min generally provides an acceptable staining intensity.
12. Immerse slides in dH2O.
13. If desired, counterstain sections with hematoxylin.
14. Wash sections in dH2O two times for 5 min each.
15. Dehydrate sections: Incubate sections in 95% ethanol two times for 10 sec each; Repeat in 100% ethanol, incubating sections two times for 10 sec each; Repeat in xylene, incubating sections two times for 10 sec each.
16. Mount sections with coverslips and mounting medium.
 

참조

  • https://pubmed.ncbi.nlm.nih.gov/12740386/
  • https://pubmed.ncbi.nlm.nih.gov/19730801/

적용 데이터

WB

Selleck 검증

  • F2333-wb
    Lane 1: Rat2 (starvation, 24 h), Lane 2: Rat2 (starvation, 24 h; EGF, 200 ng/ml, 30 min)