PI3 Kinase Class III Antibody [C6E8]

Application: Reactivity: Human, Mouse, Rat, Monkey

Lane 1: 293

Usage Information

Dilution
WB1:1000

Application
WB

Reactivity
Human, Mouse, Rat, 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
100 kDa

Positive Control	293 cells; RN33B cells
Negative Control

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)，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.

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.

Datasheet & SDS

Biological Description

Specificity
PI3 Kinase Class III Antibody [C6E8] detects endogenous levels of total PI3 Kinase Class III protein.

Subcellular Location
Cytoplasmic vesicle, Endosome

Uniprot ID
Q8NEB9

Clone
C6E8

Synonym(s)
Phosphatidylinositol 3-kinase catalytic subunit type 3; PI3-kinase type 3; PI3K type 3; PtdIns-3-kinase type 3; Phosphatidylinositol 3-kinase p100 subunit; Phosphoinositide-3-kinase class 3; hVps34; PIK3C3; VPS34

Background
PI3 kinase class III, known as VPS34 or PIK3C3, stands as the sole member of its class within the phosphoinositide 3-kinase family and acts as the mammalian counterpart to yeast Vps34. It pairs with the regulatory subunit VPS15 (p150), a serine/threonine kinase-like protein myristoylated at its N-terminus for membrane targeting. The catalytic core features a helical domain, C-terminal lipid kinase domain with conserved HRD motif for ATP binding, and an active site that phosphorylates phosphatidylinositol (PI) at the 3-position to yield PI(3)P. VPS15 stabilizes VPS34 through interactions at the N-terminal lobe, while PI(3)P production recruits FYVE and PX domain effectors like EEA1 to endosomal compartments. Cryo-EM reveals distinct complexes: PI3KC3-C1 (with VPS34-VPS15-Beclin1-ATG14L) drives autophagosome nucleation at the endoplasmic reticulum via PI(3)P-enriched omegasomes, and PI3KC3-C2 (with VPS34-VPS15-Beclin1-UVRAG-Rubicon) matures endosomes for lysosome fusion. VPS34 generates PI(3)P gradients essential for vesicular trafficking, protein sorting from early endosomes, and cytokinesis completion. In autophagy, PI(3)P scaffolds the PI3P effector complex (PEC) with WIPI2 for phagophore expansion and LC3 lipidation, coupling nutrient sensing to degradation of damaged organelles. It integrates with mTORC1 to suppress autophagy under amino acid abundance and activates it during starvation via Beclin1 dephosphorylation. Trimeric G-protein signaling modulates VPS34 through Ric-8A, linking receptor activation to membrane dynamics. Disease ties emerge in cancer, where VPS34 haploinsufficiency impairs tumor suppression; neurodegeneration, as PI(3)P deficits disrupt amyloid clearance; and immunity, with defective phagocytosis in Crohn's disease models. Overactive VPS34 fuels metastasis via endosomal trafficking of growth factor receptors, while inhibitors like wortmannin block autophagic flux in therapeutic contexts. Myristoylated VPS15 binds GTP, allosterically relieving autoinhibition to expose the VPS34 active site near membranes. These roles position PI3 kinase class III at the nexus of catabolism, trafficking, and stress adaptation.

Background

PI3 kinase class III, known as VPS34 or PIK3C3, stands as the sole member of its class within the phosphoinositide 3-kinase family and acts as the mammalian counterpart to yeast Vps34. It pairs with the regulatory subunit VPS15 (p150), a serine/threonine kinase-like protein myristoylated at its N-terminus for membrane targeting. The catalytic core features a helical domain, C-terminal lipid kinase domain with conserved HRD motif for ATP binding, and an active site that phosphorylates phosphatidylinositol (PI) at the 3-position to yield PI(3)P. VPS15 stabilizes VPS34 through interactions at the N-terminal lobe, while PI(3)P production recruits FYVE and PX domain effectors like EEA1 to endosomal compartments. Cryo-EM reveals distinct complexes: PI3KC3-C1 (with VPS34-VPS15-Beclin1-ATG14L) drives autophagosome nucleation at the endoplasmic reticulum via PI(3)P-enriched omegasomes, and PI3KC3-C2 (with VPS34-VPS15-Beclin1-UVRAG-Rubicon) matures endosomes for lysosome fusion. VPS34 generates PI(3)P gradients essential for vesicular trafficking, protein sorting from early endosomes, and cytokinesis completion. In autophagy, PI(3)P scaffolds the PI3P effector complex (PEC) with WIPI2 for phagophore expansion and LC3 lipidation, coupling nutrient sensing to degradation of damaged organelles. It integrates with mTORC1 to suppress autophagy under amino acid abundance and activates it during starvation via Beclin1 dephosphorylation. Trimeric G-protein signaling modulates VPS34 through Ric-8A, linking receptor activation to membrane dynamics. Disease ties emerge in cancer, where VPS34 haploinsufficiency impairs tumor suppression; neurodegeneration, as PI(3)P deficits disrupt amyloid clearance; and immunity, with defective phagocytosis in Crohn's disease models. Overactive VPS34 fuels metastasis via endosomal trafficking of growth factor receptors, while inhibitors like wortmannin block autophagic flux in therapeutic contexts. Myristoylated VPS15 binds GTP, allosterically relieving autoinhibition to expose the VPS34 active site near membranes. These roles position PI3 kinase class III at the nexus of catabolism, trafficking, and stress adaptation.

References
https://pubmed.ncbi.nlm.nih.gov/22308354/ https://pubmed.ncbi.nlm.nih.gov/30397185/

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%