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Western blot analysis of PKM2 on different lysates with Rabbit anti-PKM2 antibody (ER1901-90) at 1/1,000 dilution.
Lane 1: SiHa cell lysate (15 µg/Lane)
Lane 2: Mouse spleen tissue lysate (30 µg/Lane)
Predicted band size: 58 kDa
Observed band size: 58 kDa
Exposure time: 8 seconds;
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 (ER1901-90) at 1/1,000 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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☑ Knockout (KO)
All lanes: Western blot analysis of PKM with anti-PKM antibody (ER1901-90) at 1:500 dilution.
Lane 1: Wild-type MDA-MB-231 whole cell lysate.
Lane 2: PKM knockout MDA-MB-231 whole cell lysate.
ER1901-90 was shown to specifically react with PKM in wild-type MDA-MB-231 cells. No band was observed when PKM knockout samples were tested. Wild-type and PKM knockout 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 Anti-PKM antibody (ER1901-90, 1/500) and Anti-GAPDH antibody (ET1601-4, 1/10000) were used in 5% BSA at room temperature for 2 hours. Goat Anti-Rabbit IgG H&L (HRP) Secondary Antibody (HA1001) at 1:200,000 dilution was used for 1 hour at room temperature.
Cell lysate was provided by Ubigene Biosciences (Ubigene Biosciences Co., Ltd., Guangzhou, China).
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Flow cytometric analysis of PKM2 was done on F9 cells. The cells were fixed, permeabilized and stained with the primary antibody (ER1901-90, 1/50) (red). After incubation of the primary antibody at room temperature for an hour, the cells were stained with a Alexa Fluor 488-conjugated Goat anti-Rabbit IgG Secondary antibody at 1/1000 dilution for 30 minutes.Unlabelled sample was used as a control (cells without incubation with primary antibody; black).
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