Fiche technique

Description biologique

Spécificité	Phospho-Akt (Thr450) Antibody [J1B12] reconnaît les niveaux endogènes de Phospho-Akt uniquement lorsqu'il est phosphorylé sur la Thr450.
Contexte	Akt, également connue sous le nom de PKB ou Rac, est une protéine kinase pivot qui régule la survie cellulaire et l'apoptose. Elle est activée par l'insuline et divers facteurs de croissance via une voie dépendante de la PI3 kinase qui est sensible à la wortmannine. L'activation d'Akt nécessite sa liaison aux phospholipides et sa phosphorylation ultérieure sur la Thr308 dans la boucle d'activation par PDK1, ainsi que la phosphorylation sur la Ser473 dans la région C-terminale. Akt améliore la survie cellulaire en inhibant l'apoptose par la phosphorylation et l'inactivation de cibles clés, notamment Bad, les facteurs de transcription forkhead, c-Raf et la caspase-9. La phosphatase PTEN sert de régulateur négatif majeur de la cascade de signalisation PI3K/Akt. Au-delà de ses rôles dans la promotion de la survie cellulaire et de la synthèse du glycogène, Akt régule également le cycle cellulaire en empêchant la phosphorylation et la dégradation de la cycline D1 médiées par GSK-3β et en régulant à la baisse les inhibiteurs de kinases dépendantes des cyclines p27 Kip1 et p21 Waf1/Cip1. En réponse à une lésion ischémique, Akt peut être réactivée par JNK, qui phosphoryle la Thr450, une étape d'amorçage cruciale pour une phosphorylation ultérieure par la 3-phosphoinositide-dependent protein kinase-1, restaurant l'activité d'Akt.

Spécificité

Phospho-Akt (Thr450) Antibody [J1B12] reconnaît les niveaux endogènes de Phospho-Akt uniquement lorsqu'il est phosphorylé sur la Thr450.

Contexte

Akt, également connue sous le nom de PKB ou Rac, est une protéine kinase pivot qui régule la survie cellulaire et l'apoptose. Elle est activée par l'insuline et divers facteurs de croissance via une voie dépendante de la PI3 kinase qui est sensible à la wortmannine. L'activation d'Akt nécessite sa liaison aux phospholipides et sa phosphorylation ultérieure sur la Thr308 dans la boucle d'activation par PDK1, ainsi que la phosphorylation sur la Ser473 dans la région C-terminale. Akt améliore la survie cellulaire en inhibant l'apoptose par la phosphorylation et l'inactivation de cibles clés, notamment Bad, les facteurs de transcription forkhead, c-Raf et la caspase-9. La phosphatase PTEN sert de régulateur négatif majeur de la cascade de signalisation PI3K/Akt. Au-delà de ses rôles dans la promotion de la survie cellulaire et de la synthèse du glycogène, Akt régule également le cycle cellulaire en empêchant la phosphorylation et la dégradation de la cycline D1 médiées par GSK-3β et en régulant à la baisse les inhibiteurs de kinases dépendantes des cyclines p27 Kip1 et p21 Waf1/Cip1. En réponse à une lésion ischémique, Akt peut être réactivée par JNK, qui phosphoryle la Thr450, une étape d'amorçage cruciale pour une phosphorylation ultérieure par la 3-phosphoinositide-dependent protein kinase-1, restaurant l'activité d'Akt.

Informations dutilisation

Application

WB, IP

Dilution

WB	IP
1:1000	1:50

Réactivité

Human, Mouse, Rat

Source

Rabbit Monoclonal Antibody

MW

60 kDa

Tampon de stockage

PBS, pH 7.2+50% Glycerol+0.05% BSA+0.01% NaN₃

Stockage
(À partir de la date de réception)

–20°C (avoid freeze-thaw cycles), 2 years

Méthodes expérimentales
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 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, Phosphatase 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, Phosphatase 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 ( 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 1:1000), 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 811. 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.

Méthodes expérimentales

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 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, Phosphatase 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, Phosphatase 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 ( 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 1:1000), 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

811. 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.

Références

https://pubmed.ncbi.nlm.nih.gov/16962653/
https://pubmed.ncbi.nlm.nih.gov/16306447/

Données dapplication

WB

Validé par Selleck

Lane 1: LNCaP
Lane 2: LNCaP (serum-starved; Torin, 10.5 µM, 5h)
Lane 3: LNCaP (Torin, 10.5 µM, 5h)
Lane 4: LNCaP (Torin, 0.5 µM, 24h)