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Western blot analysis of DDIT3 on different lysates with Rabbit anti-DDIT3 antibody (HA750369) at 1/5,000 dilution.
Lane 1: SW480 cell lysate (20 µg/Lane)
Lane 2: HeLa cell lysate (20 µg/Lane)
Predicted band size: 19 kDa
Observed band size: 27 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 (HA750369) 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.
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☑ Knockdown (KD)
Western blot analysis of DDIT3 on different lysates with Rabbit anti-DDIT3 antibody (HA750369) at 1/1,000 dilution.
Lane 1: SW480-si NT cell lysate
Lane 2: SW480-si DDIT3 cell lysate
Lysates/proteins at 3 µg/Lane.
Predicted band size: 19 kDa
Observed band size: 25 kDa
Exposure time: 5 minutes;
4-20% SDS-PAGE gel.
ET1703-05 was shown to specifically react with DDIT3 in SW480-si NT cells. Weakened band was observed when SW480-si DDIT3 sample was tested. SW480-si NT and SW480-si DDIT3 samples were subjected to SDS-PAGE. Proteins were transferred to a PVDF membrane and blocked with 5% NFDM in TBST for 1 hour at room temperature. The primary antibody (ET1703-05, 1/1,000) and Loading control antibody (Rabbit anti-GAPDH, ET1601-4, 1/10,000) were used in 5% BSA 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.
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Western blot analysis of DDIT3 on different lysates. Proteins were transferred to a PVDF membrane and blocked with 5% BSA in PBS for 1 hour at room temperature. The primary antibody (HA750369, 1/500) was used in 5% BSA at room temperature for 2 hours. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1:200,000 dilution was used for 1 hour at room temperature.
Positive control:
Lane 1: LOVO cell lysate
Lane 2: PC-12 cell lysate
Predicted band size: 19 kDa
Observed band size: 25 kDa
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Flow cytometric analysis of DDIT3 was done on Hela cells. The cells were fixed, permeabilized and stained with the primary antibody (HA750369, 1/50) (red). After incubation of the primary antibody at room temperature for an hour, the cells were stained with a Alexa Fluor®488 conjugate-Goat anti-Rabbit IgG Secondary antibody at 1/1,000 dilution for 30 minutes.Unlabelled sample was used as a control (cells without incubation with primary antibody; black).
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