PI3 Kinase Antibody Sampler Kit
cat.: HAK21119
Product Type: Antibody Sampler Kit
Species reactivity: Human
Applications: WB
Form: Liquid
Storage condition: Store at +4℃ after thawing. Aliquot store at -20℃. Avoid repeated freeze / thaw cycles.
Storage buffer: 1*TBS (pH7.4), 0.05% BSA, 40% Glycerol. Preservative: 0.05% Sodium Azide.
Uniprot #: SwissProt: P27986 Human | P26450 Mouse | P27986 Human | P26450 Mouse | Q63787 Rat | P42336 Human | P42337 Mouse | P42338 Human | Q3U4Q1 Mouse | Q8BTI9 Mouse | Q9Z1L0 Rat | Q8NEB9 Human | Q6PF93 Mouse | O88763 Rat | P48736 Human | Q9JHG7 Mouse
Images
HAK21119_1.jpg Fig1: Western blot analysis of Phospho-PI3K p85 (Y467) + PI3K p55 (Y199) on different lysates with Rabbit anti-Phospho-PI3K p85 (Y467) + PI3K p55 (Y199) antibody (HA721672) at 1/1,000 dilution.

Lane 1: NIH/3T3 cell lysate
Lane 2: NIH/3T3 treated with 1mM sodium orthovanadate for 30 minutes cell lysate

Lysates/proteins at 30 µg/Lane.
Predicted band size: 84 kDa
Observed band size: 55/85 kDa

Exposure time: 3 minutes 10 seconds; ECL: K1801;
4-20% SDS-PAGE gel.

Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (HA721672) at 1/1,000 dilution was used in 5% NFDM/TBST at room temperature for 2 hours. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/100,000 dilution was used for 1 hour at room temperature.
HAK21119_2.jpg Fig2: Immunocytochemistry analysis of NIH/3T3 cells treated with or without 1mM Sodium orthovanadate for 30 minutes labeling Phospho-PI3K p85 (Y467) + PI3K p55 (Y199) with Rabbit anti-Phospho-PI3K p85 (Y467) + PI3K p55 (Y199) antibody (HA721672) at 1/100 dilution.

Cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 5 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Rabbit anti-Phospho-PI3K p85 (Y467) + PI3K p55 (Y199) antibody (HA721672) at 1/100 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI.

Beta tubulin (M1305-2, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor™ 594, HA1126) was used as the secondary antibody at 1/1,000 dilution.
HAK21119_3.jpg Fig3: Western blot analysis of Phospho-PI3K p85 (Y467) + PI3K p55 (Y199) on different lysates with Rabbit anti-Phospho-PI3K p85 (Y467) + PI3K p55 (Y199) antibody (HA721672) at 1/1,000 dilution.

Lane 1: 293T overexpress Src cell lysate
Lane 2: 293T overexpress Src treated with 1mM sodium orthovanadate for 30 minutes cell lysate
Lane 3: 293T overexpress Src cell lysate, the membrane treated with λpp for 1 hour
Lane 4: 293T overexpress Src treated with 1mM sodium orthovanadate for 30 minutes cell lysate, then the membrane treated with λpp for 1 hour

Lysates/proteins at 20 µg/Lane.

Predicted band size: 84 kDa
Observed band size: 55 kDa

Exposure time: 28 seconds; ECL: K1801;

4-20% SDS-PAGE gel.

Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (HA721672) at 1/1,000 dilution was used in 5% NFDM/TBST at room temperature for 2 hours. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/100,000 dilution was used for 1 hour at room temperature.
HAK21119_4.jpg Fig4: Immunohistochemical analysis of paraffin-embedded human brain tissue with Rabbit anti-PI 3 Kinase p85 alpha antibody (ET1608-70) at 1/200 dilution.

The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (ET1608-70) at 1/200 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.
HAK21119_5.jpg Fig5: Immunohistochemical analysis of paraffin-embedded human brain tissue with Rabbit anti-PI3 Kinase p110α antibody (ET1606-36) at 1/50 dilution.

The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (ET1606-36) at 1/50 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.
HAK21119_6.jpg Fig6: Immunohistochemical analysis of paraffin-embedded human liver tissue with Rabbit anti-PI3 Kinase p110α antibody (ET1606-36) at 1/50 dilution.

The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (ET1606-36) at 1/50 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.
HAK21119_7.jpg Fig7: Immunofluorescence analysis of paraffin-embedded rat colon tissue labeling PI3 Kinase p110 beta with Rabbit anti-PI3 Kinase p110 beta antibody (HA722474) at 1/50 dilution.

The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) for 2 minutes. The tissues were blocked in 10% negative goat serum for 1 hour at room temperature, washed with PBS, and then probed with the primary antibody (HA722474, green) at 1/50 dilution overnight at 4 ℃, washed with PBS. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution. Nuclei were counterstained with DAPI (blue).
HAK21119_8.jpg Fig8: Western blot analysis of VPS34 on different lysates with Rabbit anti-VPS34 antibody (HA723229) at 1/5,000 dilution.

Lane 1: HeLa cell lysate (20 µg/Lane)
Lane 2: PC-3 cell lysate (20 µg/Lane)
Lane 3: C2C12 cell lysate (20 µg/Lane)
Lane 4: C6 cell lysate (20 µg/Lane)
Lane 5: Mouse heart tissue lysate (40 µg/Lane)
Lane 6: Mouse brain tissue lysate (40 µg/Lane)
Lane 7: Rat heart tissue lysate (40 µg/Lane)

Predicted band size: 102 kDa
Observed band size: 98 kDa

Exposure time: 15 seconds; ECL: K1801;

4-20% SDS-PAGE gel.

Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (HA723229) at 1/5,000 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature.
HAK21119_9.jpg Fig9: Western blot analysis of PI 3 Kinase p85 alpha on different lysates with Rabbit anti-PI 3 Kinase p85 alpha antibody (ET1608-70) at 1/1,000 dilution.

Lane 1: Raji cell lysate
Lane 2: NIH/3T3 cell lysate
Lane 3: C6 cell lysate
Lane 4: Mouse brain tissue lysate

Lysates/proteins at 20(cell lysate),40(tissue lysate) µg/Lane.

Predicted band size: 85 kDa
Observed band size: 85 kDa

Exposure time: 2 minutes;

4-20% SDS-PAGE gel.

Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (ET1608-70) at 1/1,000 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature.
HAK21119_10.jpg Fig10: Western blot analysis of PI3 Kinase p110α on different lysates with Rabbit anti-PI3 Kinase p110α antibody (ET1606-36) at 1/1,000 dilution.

Lane 1: HepG2 cell lysate
Lane 2: Hela cell lysate
Lane 3: Jurkat cell lysate
Lane 4: NIH/3T3 cell lysate
Lane 5: mouse brain tissue lysate（20 µg/Lane）
Lane 6: mouse liver tissue lysate（20 µg/Lane）

Lysates/proteins at 10 µg/Lane.

Predicted band size: 124 kDa
Observed band size: 110 kDa

Exposure time: 3 minutes;

4-20% SDS-PAGE gel.

Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (ET1606-36) at 1/1,000 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature.
Note: All products are “FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE”.