I ran a kill curve with G418 for Vero cells and it seems that the concentration that will kill all the cells is around 1.4mg/mL. Does anyone know if this is normal for vero cells? All help appreciated. thanks
I have used the invitrogen Bac-to-Bac baculovirus expression system in Sf9 insect cells. From my experience, it worked great. The level of expression in the Sf9 cells is very high. Also, post translational modification in the cells is comparable to that of mammalian cells. I cultured the sf9 cells in suspension, however you can culture them adherently but it would be less convenient because you would need a lot of plates to get the number of cells you need.
Yes, I am using lystate from eukaryotic cells. The transfection efficiency in the cell line I am using is usually low, so my protein is probably being expressed at low levels. The western blot shows about non-specific bands in the entire lane and I can't tell if my protein is showing up or not.
I am getting a lot of non-specific bands that show up on my western blot .
I am using an anti-his monoclonal antibody that is verified to give almost no background. I am using a goat anti-mouse polycolonal for my secondary Ab.
When I do the HRP detection, I get a lot of non-specific bands that show up on all samples (including the negative control and positive control) and I can't tell if my protein of interest is one of them or not.
I am blocking with 5% dry milk and incubating the antibodies in blocking buffer as well. I am also washing the membrane pretty good. Any suggestions? thanks