Currently, the localization study for my target protein because of another protein B has to be performed using confocal. It had been confirmed using Western Blot.
1. I wanted to know if it is necessary to perform, transfection after seeding the cells on the cover glass, followed by confocal microscopy, or perform the transfection in suppose a 12-well plate and then trypsinize it and seed the cells on the cover glass?
2. How much should be the concentration of siRNA or plasmid? Should it be the same as used in Western blot or less?
3. What must be the time of incubation after media change? 48 hours or 24 hours? Because when I seed around 10,000 cells on the cover glass and perform the transfection, cell clumping occurs after 48 hours on the cover glass.
When You obtain expression of Your protein on WB?
I would do like You suggest in 1. may try even in well of 96 well plate. For confocal there is no need of plenty of cells. How many You want to scan?
You may want them quite sparsely. Tried coating glass with some protein?
2. How do You confirm expression? fluorescent tag, antibody staining? Working condicions may work again ;-)
I want to confirm the expression using fluorescently tagged antibodies. No, I didn't try coating the glass with any protein. But I will try doing it using Poly-L-Lysine.
I obtained the results for the western blot after 48 hours of transfection. How much shall I incubate for confocal microscopy?
If you are validating a knock-down for another experiment, I would use the same lentivirus incubation times and concentrations for your ICC, WB, and other experiments.
As a side note, with my current lentivirus knockdown, I saw no significant depletion of protein at 48 and 72 hours post-transduction, but did see a significant depletion at 5 days.
Depending on the half-life of your protein of interest, you may want to leave it a bit longer following transduction.
All the best,
Sam
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