![]() Briefly, the protein samples were separated by electrophoresis on a 10% SDS-polyacrylamide gel (Expedeon, San Diego, CA) for one hour at 150V. ![]() The protein samples (protein lysate and 2x sample buffer) were denatured in a 70☌ water-bath for five minutes.Įxpedeon’s Run Blue Protein Electrophoresis, Dual Run & Blot Unit instruction manual was used to run the gel and blot the proteins. Equivalent volumes of protein lysate and 2x sample buffer (4% LDS, 0.8M Triethanolamine-Cl pH 7.6, 4% Ficoll-400, 0.025% Phenol Red, 0.025% Coomassie Brilliant Blue, 2mM EDTA (Expedeon, San Diego, CA)) were mixed. The protein lysate was centrifuged at 14000 Xg for 15 minutes at 4☌ and the supernatant (protein lysate) was transferred to a new 1.5 ml tube. To measure levels of viral protein expression, cell pellets from virus infected culture were resuspended in 200μl radioimmunoprecipitation buffer (RIPA, 50mM Tris-HCL pH 7.4, 150mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) (Boston Bioproducts, Ashland, MA) including a protease inhibitor cocktail (Thermo Scientific, Waltham, MA), then incubated on ice for 30 minutes, with vortexing every 10 minutes. For more information see my Western Blot protocol below. My PI and I suspect that it is whole (non-disrupted) viral particles which have attached to the wells of the gel preventing migration down the lane.ĭoes this sound plausible? And if so, what is the best way to break up the viral particles in order to get their proteins to migrate down the gel? After visualizing my membrane, there seems to be a lot of viral protein stuck at the top of the lane (of the gel) which did not migrate down. ![]() I use the LLC-MK2 cell line for my infections. I am isolating viral (Sendai virus) proteins between 4 different virus variants at 4 different time points (1, 3, 6 & 9 hr p.i.) (the pictures above are of 1 and 3 hours only) of infection. ![]()
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