- PA1-16525 - Invitrogen Antibodies EGLN1 antibody

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of human PHD2, using Product # PA1-16525. Samples: Recombinant FLAG-His-PHD1, PHD2 and PHD3 (10 ng/lane), HeLa whole cell lysate and MEFs.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockdown of PHD2 was achieved by transfecting HeLa with PHD2 specific validated siRNAs (Silencer® select Product # s29231). Western blot analysis (Fig. a) was performed using whole cell extracts from the PHD2 knockdown cells (lane 3), non-specific scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blots were probed with PHD2 Polyclonal Antibody (Product # PA1-16525, 1 µg/mL) and Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.25 µg/mL, 1:4000 dilution). Densitometric analysis of this western blot is shown in histogram (Fig. b). Loss of signal upon siRNA mediated knock down confirms that antibody is specific to PHD2.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis was performed on whole cell extracts (30 µg lysate) of HEK 293 (Lane 1), COS-7 (Lane 2) and U-87 MG (Lane 3). The blot was probed with Anti-PHD2 Polyclonal Antibody (Product # PA1-16525, 1 µg/mL) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.25 µg/mL, 1:4000 dilution). A 46 kDa band corresponding to PHD2 was observed across the cell lines tested.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis of PHD2 in 10 ng/lane recombinant FLAG-His-PHD1, PHD2 and PHD3 , HeLa whole cell lysate and MEFs. Samples were incubated in PHD2 polyclonal antibody (Product # PA1-16525.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescence analysis of PHD2 was performed using 70% confluent log phase HeLa cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 15 minutes, and blocked with 1% BSA for 1 hour at room temperature. The cells were labeled with PHD2 Polyclonal Antibody (Product # PA1-16525) at 5 µg/mL in 0.1% BSA, incubated at 4 degree Celsius overnight and then labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034) at a dilution of 1:2000 for 45 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with SlowFade® Gold Antifade Mountant with DAPI (Product # S36938). F-actin (Panel c: red) was stained with Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image showing nucleus and cytoplasmic localization. Panel e represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Flow cytometry of PHD2 in Jurkat cells (fixed, permeabilized). Samples were incubated in PHD2 polyclonal (Product # PA1-16525) using a dilution of 3.0 µg/mL (in a 150 µL reaction).

PA1-16525