1- Magic Mark Standard
2- WtF 6hr
3- WtZ 6hr
5- F1R 6hr
6- Mock infected
7- WtF 9hr
8- WtZ 9hr
9- RGV0 9hr
10- F1R 9hr
11- Mock infected
GAPDH probe of above membrane
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. I use the LLC-MK2 cell line for my infections. 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.
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.
Does 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?
-For more information see my Western Blot protocol below.
Western Blot Protocol:
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. The protein lysate was centrifuged at 14000 Xg for 15 minutes at 4°C and the supernatant (protein lysate) was transferred to a new 1.5 ml tube. 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 samples (protein lysate and 2x sample buffer) were denatured in a 70°C water-bath for five minutes.
Expedeon’s Run Blue Protein Electrophoresis, Dual Run & Blot Unit instruction manual was used to run the gel and blot the proteins. Briefly, the protein samples were separated by electrophoresis on a 10% SDS-polyacrylamide gel (Expedeon, San Diego, CA) for one hour at 150V. A Pre-Stained protein ladder (EZ-Run Pre-Stained Rec Protein ladder, Fisher, Pittsburgh, PA) and a western protein ladder (MagicMark XP Protein Western Standard, Invitrogen, Carlsbad, CA) were run alongside the protein samples. Tris- Tricine SDS (1.2M Tris, 0.8M Tricine 2% SDS, 50mM Sodium Bisulfite pH 8.2, Expedeon, San Diego, CA) run buffer was used. Following electrophoresis, the gel was transferred onto a nitrocellulose membrane (Whatman, Piscataway, NJ) for 1.5 hours at 200V at room temperature with ice packs and a stir bar. To prevent nonspecific binding of antibody, the membrane was blocked in 5% non-fat dry milk (Bio-Rad, Hercules, CA) in TBST (Tris buffered saline (Boston Bioproducts, Ashland, MA) and 0.1% Tween). After blocking, the membrane was incubated in a 1:2000 dilution of a rabbit anti-SeV primary antibody (MBL International, Woburn, MA) solution for one hour at room temperature with rocking, followed by washing three times, five minutes each, with TBST at room temperature. To detect primary antibody attachment to SeV proteins, a 1:1500 dilution of horseradish peroxidase-conjugated anti-rabbit IgG secondary antibody solution was added to the membrane for one hour at room temperature with rocking; followed by membrane washing with TBST three times, five minutes each, at room temperature. Proteins were visualized using the Pierce™ ECL Western Blotting Substrate kit (Thermo Scientific, Rockford, IL) and analyzed using the Molecular Imager VersaDoc™ MP Imaging System with the Quantity One Analysis Software (Bio-Rad, Hercules CA).
In order to normalize the blot results, the membranes were stripped (62.5mM Tris-HCL pH 6.8, 2% SDS, 100μM β-mercaptoethanol) at 50°C for 30 minutes and then washed five times in TBST for five minutes at room temperature. The membranes were incubated overnight at 4°C in 5% non-fat dry milk to block non-specific binding sites. On the following day the membranes were incubated with a 1:1000 dilution of mouse anti-GAPDH primary antibody (Research Diagnostics Inc, Flanders, NJ) probe for one hour at room temperature with rocking. The membranes were washed three times in TBST for five minutes at room temperature with rocking and then incubated in a 1:3000 dilution of horseradish peroxidase-conjugated anti-mouse IgG secondary antibody (Amersham Life Sciences, Piscataway, NJ) for one hour at room temperature with rocking. Proteins were visualized as described above.
Thanks for reading and any help is much appreciated!