Datasheet

生物学的記述

Specificity	PDK1 Antibody [M15P23] detects endogenous levels of total PDK1 protein.
Background	PDK1, a 556-amino acid serine/threonine kinase of the AGC family, acts as a master upstream activator by phosphorylating the T-loop or activation segment of more than 20 AGC kinases, including Akt or PKB, p70S6K, RSK, SGK, and PKC isoforms, thereby integrating PI3K signaling to control cell survival, proliferation, and metabolism. PDK1 consists of an N-terminal catalytic kinase domain spanning amino acids 1 to 350, which is bilobal and contains a PIF-pocket hydrophobic cleft formed by Leu155, Val158, and Phe160 adjacent to the activation loop phosphate-binding site, where Arg129, Arg131, and Glu130 coordinate pSer241 and a sulfate mimic. The C-terminal pleckstrin homology PH domain, spanning amino acids 411 to 545, has a canonical seven-stranded beta-sandwich plus an alpha-helix 'budded' fold, with a basic pocket that binds PtdIns(3,4,5)P3 or PtdIns(3,4)P2 via Lys535 and Arg531. PDK1 is a dual-mode substrate activation: in the membrane-recruited mode, PIP3 or PIP2 binding to the PH domain, with around tenfold affinity, colocalizes PDK1 with PIP3-docked Akt, recruiting the hydrophobic motif FxxFS, TF, or Y into the PIF-pocket and inducing a conformational change that exposes the T-loop for phosphorylation at residues such as Ser473 on Akt or Thr229 on S6K1. In the cytosolic docking mode, the phosphorylated hydrophobic motif of S6K, RSK, or SGK binds the PIF-pocket allosterically, resulting in about 100-fold activation even in the absence of PIP3. PDK1 transduces growth factor signals, such as insulin and IGF1, through the PI3K to PIP3 to Akt to mTORC1 pathway, driving glucose uptake, glycogen synthesis, and protein translation, while its constitutive activity via Ser241 autophosphorylation sustains basal AGC kinase tone. PDK1 is amplified or overexpressed in cancers, including pancreas and breast, often due to 17q21 amplification, and targeted inhibitors synergize with PI3K or mTOR inhibitors to induce apoptosis in PTEN-null tumors.

Specificity

PDK1 Antibody [M15P23] detects endogenous levels of total PDK1 protein.

Background

PDK1, a 556-amino acid serine/threonine kinase of the AGC family, acts as a master upstream activator by phosphorylating the T-loop or activation segment of more than 20 AGC kinases, including Akt or PKB, p70S6K, RSK, SGK, and PKC isoforms, thereby integrating PI3K signaling to control cell survival, proliferation, and metabolism. PDK1 consists of an N-terminal catalytic kinase domain spanning amino acids 1 to 350, which is bilobal and contains a PIF-pocket hydrophobic cleft formed by Leu155, Val158, and Phe160 adjacent to the activation loop phosphate-binding site, where Arg129, Arg131, and Glu130 coordinate pSer241 and a sulfate mimic. The C-terminal pleckstrin homology PH domain, spanning amino acids 411 to 545, has a canonical seven-stranded beta-sandwich plus an alpha-helix 'budded' fold, with a basic pocket that binds PtdIns(3,4,5)P3 or PtdIns(3,4)P2 via Lys535 and Arg531. PDK1 is a dual-mode substrate activation: in the membrane-recruited mode, PIP3 or PIP2 binding to the PH domain, with around tenfold affinity, colocalizes PDK1 with PIP3-docked Akt, recruiting the hydrophobic motif FxxFS, TF, or Y into the PIF-pocket and inducing a conformational change that exposes the T-loop for phosphorylation at residues such as Ser473 on Akt or Thr229 on S6K1. In the cytosolic docking mode, the phosphorylated hydrophobic motif of S6K, RSK, or SGK binds the PIF-pocket allosterically, resulting in about 100-fold activation even in the absence of PIP3. PDK1 transduces growth factor signals, such as insulin and IGF1, through the PI3K to PIP3 to Akt to mTORC1 pathway, driving glucose uptake, glycogen synthesis, and protein translation, while its constitutive activity via Ser241 autophosphorylation sustains basal AGC kinase tone. PDK1 is amplified or overexpressed in cancers, including pancreas and breast, often due to 17q21 amplification, and targeted inhibitors synergize with PI3K or mTOR inhibitors to induce apoptosis in PTEN-null tumors.

使用情報

Application

WB

Dilution

WB

1:1000

Reactivity

Human, Mouse, Rat, Hamster, Monkey

Source

Rabbit Monoclonal Antibody

MW

58-68 kDa

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

プロトコール
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. 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) 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) 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 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.

プロトコール

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.

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) 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) 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 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.

References

https://pubmed.ncbi.nlm.nih.gov/23745598/
https://pubmed.ncbi.nlm.nih.gov/12169624/

Application Data

WB

Validated by Selleck

Lane 1: 2R75, Lane 2: MCF-7, Lane 3: COS-7, Lane 4: 3T3