All lanes: Western blot analysis of PKM with anti-PKM antibody (R1603-5) at 1:500 dilution. Lane 1: Wild-type MDA-MB-231 whole cell lysate. Lane 2: PKM knockout MDA-MB-231 whole cell lysate. R1603-5 was shown to specifically react with PKM in wild-type MDA-MB-231 cells. No band was observed when PKM knockout sample was 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 antibody (R1603-5; 1/500) and Loading control antibody (Rabbit anti-GAPDH ; ET1601-4; 1/10;000) 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. Cell lysate was provided by Ubigene Biosciences (Ubigene Biosciences Co.; Ltd.; Guangzhou; China).
Western blot analysis of GAPDH on Hela cell lysates with Rabbit anti-GAPDH antibody (ET1601-4). Hela cell lysates at 10 µg/Lane. Predicted band size: 36 kDa Observed band size: 36 kDa 12% 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 (ET1601-4) at serial dilution was used in 5% NFDM/TBST at room temperature for 2 hours. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1:300;000 dilution was used for 1 hour at room temperature.
Western blot analysis of Lactate Dehydrogenase on different lysates with Rabbit anti-Lactate Dehydrogenase antibody (ET1608-57) at 1/5;000 dilution. Lane 1: HeLa cell lysate (15 µg/Lane) Lane 2: HEK-293 cell lysate (15 µg/Lane) Lane 3: A549 cell lysate (15 µg/Lane) Predicted band size: 37 kDa Observed band size: 37 kDa Exposure time: 1 minute 36 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 (ET1608-57) 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.
