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

Catalogusnummer F2333

Afdrukken

Biologische Beschrijving

Specificiteit

Phospho-p70 S6K (Thr421/Ser424) Antibody [E5J12] detecteert endogene niveaus van p70 S6 kinase alleen wanneer gefosforyleerd op Thr421 en Ser424 respectievelijk.
Achtergrond

Phospho-p70 S6 kinase (Thr421/Ser424) verwijst naar de gefosforyleerde vorm van p70S6K op threonine 421 en serine 424, residuen die zich bevinden binnen het carboxy-terminale autoinhibitoire domein. p70S6K, ook wel ribosomal S6 kinase 1 (S6K1) genoemd, is een 70-kDa serine/threonine kinase dat behoort tot de AGC kinase familie en een belangrijke stroomafwaartse effector is van de PI3K/Akt/mTOR-route. Het volledige enzym bevat een N-terminale regulerende regio, een katalytisch kinase domein en een C-terminaal autoinhibitoir segment dat de activiteit regelt. p70S6K wordt ubiquitair tot expressie gebracht in zoogdierweefsels, met name hoge niveaus in metabolisch actieve en proliferatieve cellen, waaronder fibroblasten, immuuncellen en ontwikkelende weefsels. Fosforylering van Thr421/Ser424 vindt plaats als reactie op groeifactoren, cytokines zoals GM-CSF en mitogene signalen, en verlicht autoinhibitie om daaropvolgende fosforylering op Thr389 en Thr229 mogelijk te maken, kritieke stappen voor volledige activering. Functioneel draagt fosforylering op Thr421/Ser424 bij aan verhoogde enzymatische activiteit van p70S6K, die op zijn beurt substraten zoals ribosomaal eiwit S6 en eIF4B fosforyleert, waardoor translatie-initiatie, eiwitsynthese, celcyclusprogressie, groei en proliferatie worden bevorderd. In hematopoëtische cellen zoals neutrofielen integreert GM-CSF-geïnduceerde fosforylering op deze residuen signalen van zowel mTOR- als MAPK-afhankelijke routes, wat Phospho-Thr421/Ser424 benadrukt als een belangrijk regulerend checkpoint voor S6K-activering bij ontsteking, immuunresponsen en groeicontrole.

Gebruiksinformatie

Toepassing WB, IHC, ELISA Verdunning
WB IHC
1:500 - 1:3000 1:100
Reactiviteit Human, Mouse, Rat
Bron Rabbit Monoclonal Antibody MW 59 kDa
Opslagbuffer PBS, pH 7.2+50% Glycerol+0.05% BSA+0.01% NaN3
Opslag
(Vanaf de datum van ontvangst)		 -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.
 

Referenties

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

Toepassingsgegevens

WB

Gevalideerd door Selleck

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