Anti-PI 3 Kinase p85 alpha antibody [EPR5513]

• WB

Lab

Western blot - Anti-PI 3 Kinase p85 alpha antibody [EPR5513] (AB133595)

Lane 1 : Wild-type HAP1 cell lysate (20 μg)
Lane 2 : PI 3 Kinase p85 alpha knockout HAP1 cell lysate (20 μg)
Lane 3 : Jurkat cell lysate (20 μg)
Lane 4 : THP1 cell lysate (20 μg)
Lanes 1 - 4 : Merged signal (red and green). Green - ab133595 observed at 90 kDa. Red - loading control, ab8245, observed at 37 kDa.
ab133595 was shown to specifically react with PI 3 Kinase p85 alpha when PI 3 Kinase p85 alpha knockout samples were used. Wild-type and PI 3 Kinase p85 alpha knockout samples were subjected to SDS-PAGE. ab133595 and ab8245 (loading control to GAPDH) were diluted 1/1000 and 1/2000 respectively and incubated overnight at 4°C. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (ab216776) secondary antibodies at 1/10000 dilution for 1 h at room temperature before imaging.

All lanes:

Western blot - Anti-PI 3 Kinase p85 alpha antibody [EPR5513] (ab133595)

Predicted band size: 83 kDa

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• WB

Lab

Western blot - Anti-PI 3 Kinase p85 alpha antibody [EPR5513] (AB133595)

Lanes 1- 2 : Merged signal (red and green). Green - ab133595 observed at 90 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

ab133595 was shown to react with PI 3 Kinase p85 alpha in wild-type HeLa cells in western blot. Loss of signal was observed when knockout cell line ab265116 (knockout cell lysate ab257029) was used. Wild-type HeLa and PIK3R1 knockout HeLa cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab133595 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) overnight at 4°C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^®800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^®680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-PI 3 Kinase p85 alpha antibody [EPR5513] (ab133595) at 1/1000 dilution

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

PIK3R1 knockout HeLa cell lysate at 20 µg

Lane 2:

Western blot - Human PIK3R1 (PI 3 Kinase p85 alpha) knockout HeLa cell line (<a href='/en-us/products/cell-lines/human-pik3r1-pi-3-kinase-p85-alpha-knockout-hela-cell-line-ab265116'>ab265116</a>)

Predicted band size: 83 kDa

Observed band size: 90 kDa

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• WB

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Western blot - Anti-PI 3 Kinase p85 alpha antibody [EPR5513] (AB133595)

All lanes:

Western blot - Anti-PI 3 Kinase p85 alpha antibody [EPR5513] (ab133595) at 1/1000 dilution

Lane 1:

Jurkat cell lysate at 10 µg

Lane 2:

Raji cell lysate at 10 µg

Lane 3:

K562 cell lysate at 10 µg

Lane 4:

MCF7 cell lysate at 10 µg

Lane 5:

C6 cell lysate at 10 µg

Lane 6:

Raw 264.7 cell lysate at 10 µg

Lane 7:

PC12 cell lysate at 10 µg

Lane 8:

NIH3T3 cell lysate at 10 µg

Secondary

All lanes:

HRP labelled Goat anti Rabbit at 1/2000 dilution

Predicted band size: 83 kDa

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• OI-RD Scanning

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OI-RD Scanning - Anti-PI 3 Kinase p85 alpha antibody [EPR5513] (AB133595)

We have systematically measured KD (the equilibrium dissociation constant between the antibody and its antigen), of more than 840 recombinant antibodies to assess not only their individual KD values but also to see the average affinity of antibody. Based on the comparison with published literature values for mouse monoclonal antibodies, Recombinant antibodies appear to be on average 1-2 order of magnitude higher affinity.