- PA5-143925 - Invitrogen Antibodies DIO1 antibody

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of DIO1 using DIO1 antibody (Product # PA5-143925). Electrophoresis was performed on a 5-20% SDS-PAGE gel at 70V (Stacking gel) / 90V (Resolving gel) for 2-3 hours. The sample well of each lane was loaded with 50 µg of sample under reducing conditions. Lane 1: rat liver tissue lysates; Lane 2: rat kidney tissue lysates; Lane 3: human A549 whole cell lysates. After Electrophoresis, proteins were transferred to a Nitrocellulose membrane at 150mA for 50-90 minutes. Blocked the membrane with 5% Non-fat Milk/ TBS for 1.5 hour at RT. The membrane was incubated with rabbit anti-DIO1 antigen affinity purified polyclonal antibody (Product # PA5-143925) at 0.5 μg/mL overnight at 4°C, then washed with TBS-0. 1%Tween 3 times with 5 minutes each and probed with a goat anti-rabbit IgG-HRP secondary antibody at a dilution of 1:5,000 for 1.5 hour at RT. The signal is developed using an Enhanced Chemiluminescent detection (ECL) kit with Tanon 5200 system. A specific band was detected for DIO1 at approximately 29KD. The expected band size for DIO1 is at 29KD.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunocytochemistry analysis of DIO1 in A549 cells. Enzyme antigen retrieval was performed using IHC enzyme antigen retrieval reagent for 15 mins. The cells were blocked with 10% goat serum. Samples were then incubated in DIO1 Polyclonal antibody (Product # PA5-143925) using a dilution of 2 μg/mL. DyLight®488 Conjugated Goat Anti-Rabbit IgG was used as secondary antibody at 1:100 dilution and incubated for 30 minutes at 37°C. The section was counterstained with DAPI. Visualize using a fluorescence microscope and filter sets appropriate for the label used.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Flow cytometry analysis of DIO1 in 293T cells using DIO1 Polyclonal Antibody (Product # PA5-143925), shown in overlay histogram (blue line). To facilitate intracellular staining, cells were fixed with 4% paraformaldehyde and permeabilized with permeabilization buffer. The cells were blocked with 10% normal goat serum, and incubated with the primary antibody (1 μg/1x10^6 cells) for 30 min at 20°C. DyLight 488 conjugated goat anti-rabbit IgG (5-10 µg/1x10^6 cells) was used as secondary antibody for 30 minutes at 20°C. Isotype control antibody (Green line) was rabbit IgG (1 µg/1x10^6) used under the same conditions. Unlabelled sample without incubation with primary antibody and secondary antibody (Red line) was used as a blank control.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Flow cytometry of DIO1 in ANA-1 cells (Blue line). The cells were blocked with 10% normal goat serum, and then incubated with DIO1 polyclonal antibody (Product # PA5-143925) (1 μg/1x10^6 cells) for 30 min at 20°C. Isotype control antibody (Green line) was rabbit IgG (1 μg/1x10^6 cells) used under the same conditions. Unlabeled sample (Red line) was also used as a control. DyLight®488 conjugated goat anti-rabbit IgG (5-10 μg/1x10^6 cells) was used as secondary antibody for 30 minutes at 20°C.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Flow cytometry analysis of DIO1 in ANA-1 cells using DIO1 Polyclonal Antibody (Product # PA5-143925), shown in overlay histogram (blue line). To facilitate intracellular staining, cells were fixed with 4% paraformaldehyde and permeabilized with permeabilization buffer. The cells were blocked with 10% normal goat serum, and incubated with the primary antibody (1 μg/1x10^6 cells) for 30 min at 20°C. DyLight 488 conjugated goat anti-rabbit IgG (5-10 µg/1x10^6 cells) was used as secondary antibody for 30 minutes at 20°C. Isotype control antibody (Green line) was rabbit IgG (1 µg/1x10^6) used under the same conditions. Unlabelled sample without incubation with primary antibody and secondary antibody (Red line) was used as a blank control.

PA5-143925