Novex 4 20 tris glycine 12 well polyacrylamide gradient gels

Lysis buffer (12.5 ml Tris HCL, 2g SDS, 10 ml Glycerol, 67.5 ml Distilled Water) was used to harvest whole-cell lysates, followed by sonication. The concentration of protein in the cell lysates was estimated by using a bicinchoninic acid (BCA) assay. Novex® 4-20% Tris-Glycine 12-well polyacrylamide gradient gels (Invitrogen, UK) were used to separate proteins. The separated proteins were transferred by perpendicular electrophoresis to a nitrocellulose Hybond^TM C membrane (Amersham, Buckinghamshire, UK). Monoclonal Mouse Anti-Human primary antibodies Actin 1:1000 (#: A4700, Sigma-Aldrich), MDM2 1:300 (#: OP46-100UG, Merck Millipore), p21^WAF1 1:100 (#: OP64, Calbiochem), p53 1:500 (#: NCL-L-p53-DO7, Leica Microsystems Ltd.) and Polyclonal Rabbit Anti-Human primary antibody BAX 1:1000 (#: 2772S, Cell Signalling) were used. Secondary goat anti-mouse/rabbit HRP-conjugated antibodies (#: P0447/P0448, Dako) were used at 1:1000. All antibodies were diluted in 5% milk/1XTBS-Tween (w/v). Enhanced chemiluminescence (GE Life Sciences) and X-ray film (Fujifilm) were used to visualize the proteins.

Lysis buffer (12.5 ml Tris HCL, 2g SDS, 10 ml Glycerol, 67.5 ml Distilled Water) was used to harvest whole-cell lysates, followed by sonication. The concentration of protein in the cell lysates was estimated by using a bicinchoninic acid (BCA) assay. Novex® 4-20% Tris-Glycine 12-well polyacrylamide gradient gels (Invitrogen, UK) were used to separate proteins. The separated proteins were transferred by perpendicular electrophoresis to a nitrocellulose Hybond™ C membrane (Amersham, Buckinghamshire, UK). Monoclonal Mouse Anti-Human primary antibodies Actin 1:1000 (#: A4700, Sigma-Aldrich), MDM2 1:300 (#: OP46-100UG, Merck Millipore), p21^WAF1 1:100 (#: OP64, Calbiochem), PUMA 1:1000 (#dd716, Santa Cruz Biotechnology) and p53 1:500 (#: NCL-L-p53-DO7, Leica Microsystems Ltd.) were used. Secondary goat anti-mouse HRP-conjugated antibodies (#: P0447/P0448, Dako) were used at 1:1000. All antibodies were diluted in 5% milk/1XTBS-Tween (w/v). Enhanced chemiluminescence (GE Life Sciences) and X-ray film (Fujifilm) were used to visualize the proteins.