I'm trying to introduce a plasmid encoding for a fusion protein calreticulin-RFP but for now I have no positive expression of the plasmid info. The modification was done a week ago. Any suggestions?
thanks in advance
Hi Marc,
It is for transient expression or you are trying to make a stable line?
Do you have a parallel transfection with e.g. a GFP or ß-galatosidase gene to give you an idea of the transfection efficiency?
For 293 cells we use PEI-MAX and that gives us really nice transfection efficiencies and is not so expensive-you may have to play with PEI:DNA ratios to optimize for your cell line but it is normally very efficient.
It is, for now, for transient expression. I have also a parallel transfection done with a GFP gene.
Thanks for the suggestion!
You should really see some RFP within 24-48hrs if you have a microscope with epi- fluorescence attachments unless expression levels are very low. If not you could try a Western against your protein or the RFP fusion (or another tag if present).
What vector are you using to express the fusion? The RFP is definitely in-frame with your calreticulin etc?
I had positive results with a western. I repeated the transfection PEI-MAX and it is a total different world.
Thanks for the help!!!
A BETTER world I hope? Out of interest what transfection reagent were you using before and what DNA: PEI-MAX ratio are you currently using? For adherent Hek293 we see best results with a 1:4 ratio and 15-20 minutes pre-incubation time-this ratio/time is also useful because it is fairly insensitive to changing cell densities.
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Molecular Biological Techniques