CC membrane protein characterization

Cancer Cell Membrane (CC) Protein Characterization

1. The cracked cancer cell membrane samples were suspended in lithium dodecyl sulfate (LDS) loading buffer (Invitrogen). 
2. Samples were heated to 90 ^oC for 10 min, and 20 μL of sample was loaded into each well of a NuPAGE Novex 4-12% Bis-Tris minigel, using 3-(N-morpholino) propane sulfonic acid (MOPS) sodium dodecyl sulfate (SDS) as the running buffer (Invitrogen) in an XCell SureLock Electrophoresis System based on the manufacturer’s instructions.
3. Protein staining was accomplished using Coomassie Blue (Invitrogen) and destained in water overnight before imaging. 
4. For western blot analysis, the protein was transferred to Protran nitrocellulose membranes (Whatman) using an XCell II Blot Module (Invitrogen) in NuPAGE transfer buffer (Invitrogen) per the manufacturer’s instructions. 
5. Membranes were probed using antibodies against CD44 (clone 515; BD Biosciences), E-Cadherin (clone 36; BD Biosciences) and CD49e (BD Biosciences) followed by horseradish peroxidase HRP-conjugated anti-mouse IgG (Cell Signaling) as the secondary antibody. 

Category