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. For most researchers, we generally recommend:Ī) ab116027 for proteins between 10 and 180 kDaī) ab116028 for larger proteins up to 245 kDaĬ) ab116029 for smaller proteins down to 3.5 kDaĭ) ab116029 or ab234592 for studying smaller and larger proteins at the same time.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. When choosing the right pre-stained protein ladder, the most important choice is based on the size of your protein of interest. Prestained ladders also can use highlight bands of a different color, to make it easier to identify different size bands. Protein ladders are most convenient to use when they are supplied ready to use, with no heating, dilution, or addition of reducing agent required before loading onto the gel. Unstained protein ladders are more accurate for sizing proteins, as the dyes used in prestained ladders can slightly distort the apparent size of the protein ladder proteins on the gel. However, an unstained protein ladder can only be visualized following staining with Coomassie or a similar non-specific protein stain. Using an unstained protein ladder is very useful when you need to accurately calculate the size of your protein. calculate the approximate size of your protein by overlaying an image of the membrane with the ladder with the image generated by antibody staining. verify transfer efficiency between gel and membrane (PVDF, nylon, or nitrocellulose) monitor protein separation during SDS-polyacrylamide gel electrophoresis Using a prestained protein ladder when running a western blot helps when you need to:
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