Anti-PHD2 / prolyl hydroxylase antibody [EPR3660(B)(2)]

• IP

Lab

Immunoprecipitation - Anti-PHD2 / prolyl hydroxylase antibody [EPR3660(B)(2)] (AB133630)

ab133630 (purified) at 1 : 40 dilution (2μg) immunoprecipitating PHD2 / prolyl hydroxylase in SH-SY5Y treated with 0.1mM cobalt chloride for 8 hours whole cell lysate.
Lane 1 (input) : SH-SY5Y (Human neuroblastoma epithelial cell) treated with 0.1mM cobalt chloride for 8 hours whole cell lysate 10μg
Lane 2 (+) : ab133630 & SH-SY5Y treated with 0.1mM cobalt chloride for 8 hours whole cell lysate
Lane 3 (-) : Rabbit monoclonal IgG (ab172730) instead of ab133630 in SH-SY5Y treated with 0.1mM cobalt chloride for 8 hours whole cell lysate
For western blotting, VeriBlot for IP Detection Reagent (HRP) (ab131366) was used for detection at 1 : 1000 dilution.
Blocking and diluting buffer : 5% NFDM/TBST.

All lanes:

Immunoprecipitation - Anti-PHD2 / prolyl hydroxylase antibody [EPR3660(B)(2)] (ab133630)

Predicted band size: 46 kDa

Observed band size: 46 kDa

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• WB

Lab

Western blot - Anti-PHD2 / prolyl hydroxylase antibody [EPR3660(B)(2)] (AB133630)

Lane 1 : Wild-type HAP1 cell lysate (20 μg)
Lane 2 : PHD2 / prolyl hydroxylase knockout HAP1 cell lysate (20 μg)
Lane 3 : HeLa cell lysate (20 μg)
Lane 4 : HepG2 cell lysate (20 μg)
Lanes 1 - 4 : Merged signal (red and green). Green - ab133630 observed at 1X kDa. Red - loading control, ab8245, observed at 37 kDa.

Unpurified ab133630 was shown to specifically react with PHD2 / prolyl hydroxylase when PHD2 / prolyl hydroxylase knockout samples were used. Wild-type and PHD2 / prolyl hydroxylase knockout samples were subjected to SDS-PAGE. Unpurified ab133630 and ab8245 (loading control to GAPDH) were diluted 1/1000 and 1/10000 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 1/10 000 dilution for 1 h at room temperature before imaging.

All lanes:

Western blot - Anti-PHD2 / prolyl hydroxylase antibody [EPR3660(B)(2)] (ab133630)

Predicted band size: 46 kDa

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• WB

Lab

Western blot - Anti-PHD2 / prolyl hydroxylase antibody [EPR3660(B)(2)] (AB133630)

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

All lanes:

Western blot - Anti-PHD2 / prolyl hydroxylase antibody [EPR3660(B)(2)] (ab133630) at 1/5000 dilution

Lane 1:

SH-SY5Y (Human neuroblastoma epithelial cell) whole cell lysates at 20 µg

Lane 2:

SH-SY5Y (Human neuroblastoma epithelial cell) treated with 0.1mM cobalt chloride for 8 hours whole cell lysates at 20 µg

Lane 3:

HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysates at 20 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/2000 dilution

Predicted band size: 46 kDa

Observed band size: 46 kDa

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• OI-RD Scanning

Unknown

OI-RD Scanning - Anti-PHD2 / prolyl hydroxylase antibody [EPR3660(B)(2)] (AB133630)

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.