Phospho-Akt (Thr308) Antibody [F21G22]

Application: Reactivity: Human, Mouse, Rat, Hamster, Monkey

Lane 1: NIH/3T3, Lane 2: NIH/3T3 (PDGF-treated), Lane 3: Jurkat, Lane 4: Jurkat (LY294002-treated)

Usage Information

Dilution
WB1:1000

Application
WB

Reactivity
Human, Mouse, Rat, Hamster, Monkey

Source
Rabbit Monoclonal Antibody

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

Storage (from the date of receipt)
-20°C (avoid freeze-thaw cycles), 2 years

Predicted MW
60 kDa

Positive Control	NIH/3T3 cells (PDGF-treated); Jurkat cells
Negative Control	Jurkat cells (LY294002-treated); NIH/3T3 cells

Experimental Methods

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. 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 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.

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.

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

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.

Datasheet & SDS

Biological Description

Specificity
Phospho-Akt (Thr308) Antibody [F21G22] detects endogenous levels of total Akt protein only when it is phosphorylated at Thr308.

Subcellular Location
Cell membrane, Cytoplasm, Membrane, Mitochondrion, Nucleus

Uniprot ID
P31749

Clone
F21G22

Synonym(s)
AKT1; AKT2; AKT3; AKT serine/threonine kinase 1; v‑akt murine thymoma viral oncogene homolog 1; C‑AKT; PRKBA; RAC‑PKα; AKT serine/threonine kinase 2; RAC‑β; PRKBB; AKT serine/threonine kinase 3; PRKBG; STK‑2

Background
Phospho-Akt (Thr308) marks the key activated form of Akt (PKB or Rac), a serine/threonine kinase in the AGC family that transduces signals from insulin, growth factors, and survival cues through the PI3K pathway to orchestrate cell fate. Akt comprises an N-terminal pleckstrin homology (PH) domain binding PIP3 for membrane localization, a central kinase domain featuring the activation loop residue Thr308 phosphorylated by PDK1, and a C-terminal domain with Ser473 modified by mTORC2 (containing rictor and Sin1) for full activation. This dual phosphorylation at Thr308 and Ser473 unleashes Akt’s kinase activity after phospholipid docking. Akt primarily blocks apoptosis by phosphorylating pro-death factors, Bad to sequester it from Bcl-2, caspase-9 to halt executioner activity, forkhead transcription factors (FoxO) to prevent pro-apoptotic gene expression, and c-Raf to fine-tune survival cascades. Akt inhibits GSK-3α/β, activating glycogen synthase for glucose storage and enhancing GLUT4 translocation for insulin-driven uptake. Cell cycle progression relies on Akt stabilizing cyclin D1 against GSK-3β degradation and exporting CDK inhibitors p27Kip1 and p21Waf1/Cip1 from the nucleus. Growth signaling flows through Akt’s inactivation of TSC2 (tuberin), freeing Rheb to stimulate mTORC1-raptor for ribosomal biogenesis and protein synthesis. Dysregulated phospho-Akt drives tumorigenesis via PTEN loss or PI3K hyperactivation, fueling unchecked proliferation in cancers; it also underlies insulin resistance in diabetes and pathological hypertrophy in heart disease.

Background

Phospho-Akt (Thr308) marks the key activated form of Akt (PKB or Rac), a serine/threonine kinase in the AGC family that transduces signals from insulin, growth factors, and survival cues through the PI3K pathway to orchestrate cell fate. Akt comprises an N-terminal pleckstrin homology (PH) domain binding PIP3 for membrane localization, a central kinase domain featuring the activation loop residue Thr308 phosphorylated by PDK1, and a C-terminal domain with Ser473 modified by mTORC2 (containing rictor and Sin1) for full activation. This dual phosphorylation at Thr308 and Ser473 unleashes Akt’s kinase activity after phospholipid docking. Akt primarily blocks apoptosis by phosphorylating pro-death factors, Bad to sequester it from Bcl-2, caspase-9 to halt executioner activity, forkhead transcription factors (FoxO) to prevent pro-apoptotic gene expression, and c-Raf to fine-tune survival cascades. Akt inhibits GSK-3α/β, activating glycogen synthase for glucose storage and enhancing GLUT4 translocation for insulin-driven uptake. Cell cycle progression relies on Akt stabilizing cyclin D1 against GSK-3β degradation and exporting CDK inhibitors p27Kip1 and p21Waf1/Cip1 from the nucleus. Growth signaling flows through Akt’s inactivation of TSC2 (tuberin), freeing Rheb to stimulate mTORC1-raptor for ribosomal biogenesis and protein synthesis. Dysregulated phospho-Akt drives tumorigenesis via PTEN loss or PI3K hyperactivation, fueling unchecked proliferation in cancers; it also underlies insulin resistance in diabetes and pathological hypertrophy in heart disease.

References
https://pubmed.ncbi.nlm.nih.gov/24352480/

Protein Size (kDa)	PVDF Transfer Membrane Pore Size (μm)
< 10	0.22
10-20	0.22
20-30	0.45
30-45	0.45
45-70	0.45
80-100	0.45
100-140	0.45
> 140	0.45

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Protein Size (kDa)	Separating Gel Percentage
4-40	20%
12-45	15%
10-70	12.5%
15-100	10%
25-100	8%
60-210	5%